Nanoparticles for protein drug delivery

ABSTRACT

The invention discloses the nanoparticles composed of chitosan, poly-glutamic acid, and at least one protein drug or bioactive agent characterized with a positive surface charge and their enhanced permeability for paracellular protein drug and bioactive agent delivery.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 12/151,230, filed May 5, 2008, now U.S. Pat. No.7,541,046 which is a continuation-in-part application of U.S. patentapplication Ser. No. 11/398,145, filed Apr. 5, 2006, now U.S. Pat. No.7,381,716, issued Jun. 3, 2008, which is a continuation-in-partapplication of U.S. patent application Ser. No. 11/284,734, filed Nov.21, 2005, now U.S. Pat. No. 7,282,194, issued Oct. 16, 2007, which is acontinuation-in-part application of U.S. patent application Ser. No.11/029,082, filed Jan. 4, 2005, now U.S. Pat. No. 7,265,090, issued Sep.4, 2007, the entire contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention is related to medical uses of nanoparticles havinga pharmaceutical composition of chitosan and polyglutamic acid withbioactive agents and their enhanced permeability for paracellulardelivery.

BACKGROUND OF THE INVENTION

Production of pharmaceutically active peptides and proteins in largequantities has become feasible (Biomacromolecules 2004; 5:1917-1925).The oral route is considered the most convenient way of drugadministrations for patients. Nevertheless, the intestinal epithelium isa major barrier to the absorption of hydrophilic drugs such as peptidesand proteins (J. Control. Release 1996; 39:131-138). This is becausehydrophilic drugs cannot easily diffuse across the cells through thelipid-bilayer cell membranes. Attentions have been given to improvingparacellular transport of hydrophilic drugs (J. Control. Release 1998;51:35-46). The transport of hydrophilic molecules via the paracellularpathway is, however, severely restricted by the presence of tightjunctions that are located at the luminal aspect of adjacent epithelialcells (Annu. Rev. Nutr. 1995; 15:35-55). These tight junctions form abarrier that limits the paracellular diffusion of hydrophilic molecules.The structure and function of tight junctions is described, inter alia,in Ann. Rev. Physiol. 1998; 60:121-160 and in Ballard T S et al., Annu.Rev. Nutr. 1995; 15:35-55. Tight junctions do not form a rigid barrierbut play an important role in the diffusion through the intestinalepithelium from lumen to bloodstream and vice versa.

Movement of solutes between cells, through the tight junctions that bindcells together into a layer as with the epithelial cells of thegastrointestinal tract, is termed paracellular transport. Paracellulartransport is passive. Paracellular transport depends on electrochemicalgradients generated by transcellular transport and on solvent dragthrough tight junctions. Tight junctions form an intercellular barrierthat separates the apical and basolateral fluid compartments of a celllayer. Movement of a solute through a tight junction from apical tobasolateral compartments depends on the “tightness” of the tightjunction for that solute.

Polymeric nanoparticles have been widely investigated as carriers fordrug delivery (Biomaterials 2002; 23:3193-3201). Much attention has beengiven to the nanoparticles made of synthetic biodegradable polymers suchas poly-G-caprolactone and polylactide due to their goodbiocompatibility (J. Drug Delivery 2000; 7:215-232; Eur. J. Pharm.Biopharm. 1995; 41:19-25). However, these nanoparticles are not idealcarriers for hydrophilic drugs because of their hydrophobic property.Some aspects of the invention relate to a novel nanoparticle system,composed of hydrophilic chitosan and poly(glutamic acid) hydrogels thatis prepared by a simple ionic-gelation method. This technique ispromising as the nanoparticles are prepared under mild conditionswithout using harmful solvents. It is known that organic solvents maycause degradation of peptide or protein drugs that are unstable andsensitive to their environments (J. Control. Release 2001; 73:279-291).

Following the oral drug delivery route, protein drugs are readilydegraded by the low pH of gastric medium in the stomach. The absorptionof protein drugs following oral administration is challenging due totheir high molecular weight, hydrophilicity, and susceptibility toenzymatic inactivation. Protein drugs at the intestinal epithelium couldnot partition into the hydrophobic membrane and thus can only traversethe epithelial barrier via the paracellular pathway. However, the tightjunction forms a barrier that limits the paracellular diffusion ofhydrophilic molecules.

Chitosan (CS), a cationic polysaccharide, is generally derived fromchitin by alkaline deacetylation (J. Control. Release 2004; 96:285-300).It was reported from literature that CS is non-toxic and soft-tissuecompatible (Biomacromolecules 2004; 5:1917-1925; Biomacromolecules 2004;5:828-833). Additionally, it is known that CS has a special feature ofadhering to the mucosal surface and transiently opening the tightjunctions between epithelial cells (Pharm. Res. 1994; 11:1358-1361).Most commercially available CSs have a quite large molecular weight (MW)and need to be dissolved in an acetic acid solution at a pH value ofapproximately 4.0 or lower that is sometimes impractical. However, thereare potential applications of CS in which a low MW would be essential.Given a low MW, the polycationic characteristic of CS can be usedtogether with a good solubility at a pH value close to physiologicalranges (Eur. J. Pharm. Biopharm. 2004; 57:101-105). Loading of peptideor protein drugs at physiological pH ranges would preserve theirbioactivity. On this basis, a low-MW CS, obtained by depolymerizing acommercially available CS using cellulase, is disclosed herein toprepare nanoparticles of the present invention.

The γ-PGA, an anionic peptide, is a natural compound produced ascapsular substance or as slime by members of the genus Bacillus (Crit.Rev. Biotechnol. 2001; 21:219-232). γ-PGA is unique in that it iscomposed of naturally occurring L-glutamic acid linked together throughamide bonds. It was reported from literature that this naturallyoccurring γ-PGA is a water-soluble, biodegradable, and non-toxicpolymer. A related, but structurally different polymer, [poly(o-glutamicacid), α-PGA] has been used for drug delivery (Adv. Drug Deliver. Rev.2002; 54:695-713; Cancer Res. 1998; 58:2404-2409). α-PGA is usuallysynthesized from poly(γ-benzyl-L-glutamate) by removing the benzylprotecting group with the use of hydrogen bromide. Hashida et al. usedα-PGA as a polymeric backbone and galactose moiety as a ligand to targethepatocytes (J. Control. Release 1999; 62:253-262). Their in vivoresults indicated that the galactosylated α-PGA had a remarkabletargeting ability to hepatocytes and degradation of α-PGA was observedin the liver.

Thanou et al. reported chitosan and its derivatives as intestinalabsorption enhancers (Adv Drug Deliv Rev 2001; 50:S91-S101). Chitosan,when protonated at an acidic pH, is able to increase the paracellularpermeability of peptide drugs across mucosal epithelia.Co-administration of chitosan or trimethyl chitosan chloride withpeptide drugs were found to substantially increase the bioavailabilityof the peptide in animals compared with administrations without thechitosan component.

SUMMARY OF THE INVENTION

It is one object of the present invention to provide a novelnanoparticle system and methods of preparation for paracellulartransport drug delivery using a simple and mild ionic-gelation methodupon addition of a poly-γ-glutamic acid (γ-PGA) solution into regularmolecular weight chitosan solution. In one embodiment, the chitosanemployed is N-trimethyl chitosan (TMC). In an alternate embodiment, thechitosan employed is low molecular weight chitosan (low-MW CS). In oneembodiment, the molecular weight of a low-MW CS of the present inventionis about 80 kDa or less, preferably at about 40 kDa, adapted foradequate solubility at a pH that maintains the bioactivity of proteinand peptide drugs. It is stipulated that a chitosan particle with about30-50 kDa molecular weight is kidney inert. The particle size and thezeta potential value of the prepared nanoparticles are controlled bytheir constituted compositions. The results obtained by the TEM(transmission electron microscopy) and AFM (atomic force microscopy)examinations showed that the morphology of the prepared nanoparticleswas generally spherical or spheroidal in shape.

Evaluation of the prepared nanoparticles in enhancing intestinalparacellular transport was investigated in vitro in Caco-2 cellmonolayers. Some aspects of the present invention provide thenanoparticles with CS dominated on the surfaces to effectively reducethe transepithelial electrical resistance (TEER) of Caco-2 cellmonolayers. The confocal laser scanning microscopy (CLSM) observationsconfirm that the nanoparticles with CS dominated on the surface are ableto open the tight junctions between Caco-2 cells and allows transport ofthe nanoparticles via the paracellular pathways.

Some aspects of the invention relate to a method of enhancing intestinalor blood brain paracellular transport configured for delivering at leastone bioactive agent in a patient comprising administering nanoparticlescomposed of γ-PGA and chitosan, wherein the step of administering thenanoparticles may be via oral administration or injection into a bloodvessel. In one embodiment, the chitosan dominates on a surface of thenanoparticles as shell substrate and the negatively charged γ-PGA ascore substrate. In another embodiment, a substantial surface of thenanoparticles is characterized with a positive surface charge. In afurther embodiment, the nanoparticles of the present invention compriseat least one positively charged shell substrate and at least onenegatively charged core substrate. In one embodiment, all of thenegatively charged core substrate conjugates with a portion of thepositively charged shell substrate that is in the core portion so tomaintain a zero-charge (neutral) core. In one embodiment, at least onebioactive or protein drug is conjugated with the negatively charged coresubstrate or the zero-charge (neutral) core.

In a further embodiment, the chitosan of the nanoparticles is a lowmolecular weight chitosan, wherein the low molecular weight chitosan hasa molecular weight of about 50 kDa, preferably having a molecular weightof less than about 40 kDa.

In a further embodiment, the nanoparticles have a mean particle sizebetween about 50 and 400 nanometers, preferably between about 100 and300 nanometers, and most preferably between about 100 and 200nanometers.

In some embodiments, the nanoparticles are loaded with a therapeuticallyeffective amount of at least one bioactive agent, wherein the bioactiveagent is selected from the group consisting of proteins, peptides,nucleosides, nucleotides, antiviral agents, antineoplastic agents,antibiotics, and anti-inflammatory drugs.

Further, the bioactive agent may be selected from the group consistingof calcitonin, cyclosporin, insulin, oxytocin, tyrosine, enkephalin,tyrotropin releasing hormone, follicle stimulating hormone, luteinizinghormone, vasopressin and vasopressin analogs, catalase, superoxidedismutase, interleukin-II, interferon, colony stimulating factor, tumornecrosis factor and melanocyte-stimulating hormone. In one preferredembodiment, the bioactive agent is an Alzheimer antagonist.

Some aspects of the invention relate to an oral dose of nanoparticlesthat effectively enhance intestinal or blood brain paracellulartransport comprising γ-PGA or α-PGA and low molecular weight chitosan,wherein the chitosan dominates on a surface of the nanoparticles. Someaspects of the invention relate to an oral dose of nanoparticles thateffectively enhance intestinal or blood brain paracellular transportcomprising a negative component, such as γ-PGA, α-PGA, heparin, orheparan sulfate, in the core and low molecular weight chitosan, whereinthe chitosan dominates on a surface of the nanoparticles with positivecharges.

In a further embodiment, the nanoparticles comprise at least onebioactive agent, such as insulin, insulin analog, Alzheimer's diseaseantagonist, Parkison's disease antagonist, or other protein/peptide. Thebioactive agent for treating Alzheimer's disease may include memantinehydrochloride (Axurag by Merz Pharmaceuticals), donepezil hydrochloride(Aricept® by Eisai Co. Ltd.), rivastigmine tartrate (Exelon® byNovartis), galantamine hydrochloride (Reminyl® by Johnson & Johnson),and tacrine hydrochloride (Cognex® by Parke Davis). Examples of insulinor insulin analog products include, but not limited to, Humulin® (by EliLilly), Humalog® (by Eli Lilly) and Lantus® (by Aventis).

Some aspects of the invention relate to an oral dose of nanoparticlesthat effectively enhance intestinal or blood brain paracellulartransport comprising γ-PGA and low molecular weight chitosan, whereinthe nanoparticles are crosslinked with a crosslinking agent or withlight, such as ultraviolet irradiation.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, each nanoparticle comprising a first component of at leastone bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle. In oneembodiment, the third component is γ-PGA, α-PGA, derivatives or salts ofPGA, heparin or alginate. In another embodiment, the first componentcomprises insulin at a concentration range of 0.075 to 0.091 mg/ml, thesecond component at a concentration range of 0.67 to 0.83 mg/ml, and thethird component comprises γ-PGA at a concentration range of 0.150 to0.184 mg/ml.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, each nanoparticle comprising a first component of at leastone bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle, wherein theat least one bioactive agent is an antagonist for Alzheimer's disease oris for treating Alzheimer's disease selected from the group consistingof memantine hydrochloride, donepezil hydrochloride, rivastigminetartrate, galantamine hydrochloride, and tacrine hydrochloride. In afurther embodiment, the at least one bioactive agent is insulin orinsulin analog. In still another embodiment, the at least one bioactiveagent is selected from the group consisting of proteins, peptides,nucleosides, nucleotides, antiviral agents, antineoplastic agents,antibiotics, and anti-inflammatory drugs.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, wherein the nanoparticles are further encapsulated in acapsule or hard-cap capsule. In one embodiment, the nanoparticles arefreeze-dried.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, each nanoparticle comprising a first component of at leastone bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle, wherein thesecond component is crosslinked. In one embodiment, the degree ofcrosslinking is less than 50%. In another embodiment, the degree ofcrosslinking is ranged between 1% and 20%.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, each nanoparticle comprising a first component of at leastone bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle, wherein thesecond component is crosslinked with a crosslinking agent selected fromthe group consisting of genipin, its derivatives, analog, stereoisomersand mixtures thereof. In one embodiment, the crosslinking agent isselected from the group consisting of epoxy compounds, dialdehydestarch, glutaraldehyde, formaldehyde, dimethyl suberimidate,carbodiimides, succinimidyls, diisocyanates, acyl azide, reuterin,ultraviolet irradiation, dehydrothermal treatment,tris(hydroxymethyl)phosphine, ascorbate-copper, glucose-lysine andphoto-oxidizers.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, wherein the low molecule weight chitosan has a molecularweight of 80 kDa or less. In one embodiment, the low molecule weightchitosan is further grafted with a polymer having a chemical formula as:

Some aspects of the invention provide a method of enhancing intestinalor brain blood paracellular transport comprising administering a dose ofnanoparticles, wherein each nanoparticle comprises a first component ofat least one bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle. In oneembodiment, the step of administering the dose of nanoparticles is viaoral administration for enhancing intestinal paracellular transport. Inanother embodiment, the step of administering the dose of nanoparticlesis via venous administration or injection to a blood vessel forenhancing brain blood paracellular transport or reducing the blood-brainbarrier (BBB).

Some aspects of the invention provide a method of treating diabetes of apatient comprising orally administering insulin containing nanoparticleswith a dosage effective amount of the insulin to treat the diabetes,wherein at least a portion of the nanoparticles comprises a positivelycharged shell substrate and a negatively charged core substrate. In oneembodiment, the shell substrate comprises chitosan, chitin, chitosanoligosaccharides, and chitosan derivatives thereof, wherein asubstantial portion of a surface of the nanoparticles is characterizedwith a positive surface charge. In another embodiment, the coresubstrate is selected from the group consisting of γ-PGA, α-PGA,water-soluble salts of PGA, metal salts of PGA, heparin, heparinanalogs, low molecular weight heparin, glycosaminoglycans, and alginate.The molecular formula of the insulin is selected from the groupconsisting of C₂₅₄H₃₇₇N₆₅O₇₅S₆, C₂₅₇H₃₈₃N₆₅O₇₇S₆, C₂₅₆H₃₈N₆₅O₇₉S₆,C₂₆₇H₄₀₄N₇₂O₇₈S₆, C₂₆₇H₄₀₈N₇₂O₇₇S₆ (insulin glargine), C₂₆₇H₄O₂N₆₄O₇₆S₆(insulin determir), and the like.

In one embodiment, the orally administering insulin containingnanoparticles comprise a dosage effective amount of the insulin to treatthe diabetes comprising an insulin amount of between about 15 units to45 units, preferably between about 25 units to 35 units, per kilogrambody weight of the patient. In a further embodiment, theinsulin-containing nanoparticle comprises a trace amount of zinc orcalcium, or is treated with enteric coating.

In one embodiment, the insulin containing nanoparticles further compriseat least one paracellular transport enhancer, wherein the paracellulartransport enhancer may be selected from the group consisting of Ca²⁺chelators, bile salts, anionic surfactants, medium-chain fatty acids,and phosphate esters. In another embodiment, the nanoparticles and theparacellular transport enhancer are co-encapsulated in a capsule or areencapsulated separately.

Some aspects of the invention provide nanoparticles for oraladministration in a patient, comprising a positively charged shellsubstrate, a negatively charged core substrate, and a bioactive agentconjugated with the core substrate, wherein the core substrate isselected from the group consisting of heparin, heparin analogs, lowmolecular weight heparin, glycosaminoglycans, and alginate, thebioactive agent being selected from the group consisting of chondroitinsulfate, hyaluronic acid, growth factor and protein withpharmaceutically effective amount.

Some aspects of the invention provide nanoparticles for oraladministration in a patient, comprising a positively charged shellsubstrate, a negatively charged core substrate, and a bioactive agentconjugated with the core substrate, wherein the bioactive agent iscalcitonin or vancomycin.

Some aspects of the invention provide a method of treating Alzheimer'sdiseases of a patient comprising intravenously administering bioactivenanoparticles with a dosage effective to treat the Alzheimer's diseases,wherein the bioactive nanoparticles comprises a positively charged shellsubstrate, a negatively charged core substrate, and at least onebioactive agent for treating Alzheimer's disease, wherein the at leastone bioactive agent is selected from the group consisting of memantinehydrochloride, donepezil hydrochloride, rivastigmine tartrate,galantamine hydrochloride, and tacrine hydrochloride.

In one embodiment, the dosage effective to treat the Alzheimer'sdiseases comprises administering the at least one bioactive agent fortreating Alzheimer's disease at about 10 mg to 40 mg per day over aperiod of one month to one year. In another embodiment, at least aportion the shell substrate is crosslinked, preferably at a degree ofcrosslinking less than about 50%, or most preferably between about 1%and 20%.

One aspect of the invention provides a pharmaceutical composition ofnanoparticles, wherein the nanoparticles may be freeze dried to formsolid dried nanoparticles. The dried nanoparticles may be loaded in acapsule (such as a two-part hard gelatin capsule) for oraladministration in a patient, wherein the capsule may be furtherenterically coated. The freeze-dried nanoparticles can be rehydrated insolution or by contacting fluid so to revert to wet nanoparticles havingpositive surface charge. In one embodiment, nanoparticles may be mixedwith trehalose or with hexan-1,2,3,4,5,6-hexyl in a freeze-dryingprocess.

Some aspects of the invention provide a pharmaceutical composition ofnanoparticles characterized by enhancing paracellular transport, eachnanoparticle comprising a shell component and a core component, whereinat least a portion of the shell component comprises chitosan and whereinthe core component is comprised of MgSO₄, sodium tripolyphosphate, atleast one bioactive agent, and a negatively charged compound, wherein asubstantial portion of the negatively charged compound is conjugated tothe chitosan. In one embodiment, the negatively charged component of thepharmaceutical composition is γ-PGA or a derivative or salt of PGAs.

Some aspects of the invention provide an orally deliverable capsule toan animal subject comprising: (a) an empty capsule; and (b) bioactivenanoparticles loaded within the empty capsule, wherein the nanoparticlescomprise a shell substrate of chitosan, a negatively charged coresubstrate, and at least one bioactive agent. In one embodiment, theempty capsule comprises a two-part hard gelatin capsule. In anotherembodiment, the capsule is treated with enteric coating.

One object of the present invention is to provide a method ofmanufacturing the orally deliverable capsule, the method comprisingsteps of: (a) providing an empty capsule; (b) providing bioactivenanoparticles, wherein the nanoparticles comprise a shell substrate ofchitosan, a negatively charged core substrate, and at least onebioactive agent; (c) freeze-drying the nanoparticles; and (d) fillingthe freeze-dried bioactive nanoparticles into the empty capsule, therebyproducing an orally deliverable capsule. In one embodiment, thebioactive nanoparticles further comprise magnesium sulfate and TPP.

BRIEF DESCRIPTION OF THE DRAWINGS

Additional objects and features of the present invention will becomemore apparent and the disclosure itself will be best understood from thefollowing Detailed Description of the Exemplary Embodiments, when readwith reference to the accompanying drawings.

FIG. 1 shows GPC chromatograms of (a) standard-MW CS beforedepolymerization and the low-MW CS after depolymerization; (b) thepurified γ-PGA obtained from microbial fermentation.

FIG. 2 shows (a) FT-IR and (b) ¹H-NMR spectra of the purified γ-PGAobtained from microbial fermentation.

FIG. 3 shows FT-IR spectra of the low-MW CS and the prepared CS-γ-PGAnanoparticles.

FIG. 4 shows (a) a TEM micrograph of the prepared CS-γ-PGA nanoparticles(0.10% γ-PGA:0.20% CS) and (b) an AFM micrograph of the preparedCS-γ-PGA nanoparticles (0.01% γ-PGA:0.01% CS).

FIG. 5 shows changes in particle size and zeta potential of (a) theCS-γ-PGA nanoparticles (0.10% γ-PGA:0.20% CS) and (b) the CS-γ-PGAnanoparticles (0.10% γ-PGA:0.01% CS) during storage for up to 6 weeks.

FIG. 6 shows effects of the prepared CS-7-PGA nanoparticles on the TEERvalues of Caco-2 cell monolayers.

FIG. 7 shows fluorescence images (taken by an inversed confocal laserscanning microscope) of 4 optical sections of a Caco-2 cell monolayerthat had been incubated with the fCS-γ-PGA nanoparticles with a positivesurface charge (0.10% γ-PGA:0.20% CS) for (a) 20 min and (b) 60 min.

FIG. 8 shows an illustrative protein transport mechanism through a celllayer, including transcellular transport and paracelluler transport.

FIG. 9A-C shows a schematic illustration of a paracellular transportmechanism.

FIG. 10 shows an fCS-γ-PGA nanoparticle with FITC-labeled chitosanshaving positive surface charge.

FIG. 11 shows loading capacity and association efficiency of insulin innanoparticles of chitosan and γ-PGA.

FIG. 12 shows loading capacity and association efficiency of insulin innanoparticles of chitosan as reference.

FIG. 13 shows the stability of insulin-loaded nanoparticles.

FIG. 14 shows a representative in vitro study with insulin drug releaseprofile in a pH-adjusted solution.

FIG. 15 shows the effect of insulin of orally administeredinsulin-loaded nanoparticles on hypoglycemia in diabetic rats.

FIG. 16A-C shows a proposed mechanism of nanoparticles released from theenteric coated capsules.

FIG. 17 shows the schematic illustration of insulin conjugated withhistidine and/or glutamic acid side groups of the γ-PGA via zinc.

FIG. 18 shows the schematic illustration of insulin conjugated with acarboxyl side group of the γ-PGA via zinc.

FIG. 19 shows the hypoglycemia of orally administered insulin-loadednanoparticles in diabetic rats, wherein the freeze-dried nanoparticleswere loaded in an enterically coated capsule upon delivery.

FIG. 20 shows insulin-loaded nanoparticles with a core compositionconsisted of γ-PGA, MgSO₄, sodium tripolyphosphate (TPP), and insulin.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

The preferred embodiments of the present invention described belowrelate particularly to preparation of nanoparticles composed ofchitosan/poly-glutamic acid/insulin and their permeability to enhancethe intestinal or blood brain paracellular permeation by opening thetight junctions between epithelial cells. While the description setsforth various embodiment specific details, it will be appreciated thatthe description is illustrative only and should not be construed in anyway as limiting the invention. Furthermore, various applications of theinvention, and modifications thereto, which may occur to those who areskilled in the art, are also encompassed by the general conceptsdescribed below.

γ-PGA is a naturally occurring anionic homo-polyamide that is made ofL-glutamic acid units connected by amide linkages between α-amino andγ-carboxylic acid groups (Crit. Rev. Biotechnol. 2001; 21:219-232). Itis an exocellular polymer of certain Bacillus species that is producedwithin cells via the TCA cycle and is freely excreted into thefermentation broth. Its exact biological role is not fully known,although it is likely that γ-PGA is linked to increasing the survival ofproducing strains when exposed to environmental stresses. Because of itswater-solubility, biodegradability, edibility, and non-toxicity towardhumans and the environment, several applications of γ-PGA in food,cosmetics, medicine, and water treatment have been investigated in thepast few years.

Example No. 1 Materials and Methods of Nanoparticles Preparation

CS (MW ˜2.8×10⁵) with a degree of deacetylation of approximately 85% wasacquired from Challenge Bioproducts Co. (Taichung, Taiwan). Acetic acid,cellulase (1.92 units/mg), fluorescein isothiocyanate (FITC), phosphatebuffered saline (PBS), periodic acid, sodium acetate, formaldehyde,bismuth subnitrate, and Hanks balanced salt solution (HBSS) werepurchased from Sigma Chemical Co. (St. Louis, Mo.). Ethanol absoluteanhydrous and potassium sodium tartrate were obtained from Merck(Darmstadt, Germany). Non-essential amino acid (NEAA) solution, fetalbovine serum (FBS), gentamicin and trypsin-EDTA were acquired from Gibco(Grand Island, N.Y.). Eagle's minimal essential medium (MEM) waspurchased from Bio West (Nuaille, France). All other chemicals andreagents used were of analytical grade.

Example No. 2 Depolymerization of CS by Enzymatic Hydrolysis

Regular CS was treated with enzyme (cellulase) to produce low-MW CSaccording to a method described by Qin et al. with some modifications(Food Chem. 2004; 84:107-115). A solution of CS (20 g/l) was prepared bydissolving CS in 2% acetic acid. Care was taken to ensure totalsolubility of CS. Then, the CS solution was introduced into a vessel andadjusted to the desired pH 5.0 with 2N aqueous NaOH. Subsequently,cellulase (0.1 g) was added into the CS solution (100 ml) andcontinuously stirred at 37° C. for 12 hours. Afterward, thedepolymerized CS was precipitated with aqueous NaOH at pH 7.0-7.2 andthe precipitated CS was washed three times with deionized water. Theresulting low-MW CS was lyophilized in a freeze dryer (Eyela Co. Ltd,Tokyo, Japan).

The average molecular weight of the depolymerized CS was determined by agel permeation chromatography (GPC) system equipped with a series of PLaquagel-OH columns (one Guard 8 μm, 50×7.5 mm and two MIXED 8 μm,300×7.5 mm, PL Laboratories, UK) and a refractive index (RI) detector(RI2000-F, SFD, Torrance, Calif.). Polysaccharide standards (molecularweights range from 180 to 788,000, Polymer Laboratories, UK) were usedto construct a calibration curve. The mobile phase contained 0.01MNaH₂PO₄ and 0.5M NaNO₃ and was brought to a pH of 2.0. The flow rate ofmobile phase was 1.0 ml/min, and the columns and the RI detector cellwere maintained at 30° C.

Factors limiting applications of most commercially available CSs aretheir high molecular weight and thus high viscosity and poor solubilityat physiological pH ranges. Low-MW CS overcomes these limitations andhence finds much wider applications in diversified fields. It wassuggested that low-MW CS be used as a parenteral drug carrier due to itslower antigen effect (Eur. J. Pharm. Biopharm. 2004; 57:101-105). Low-MWCS was used as a non-viral gene delivery system and showed promisingresults (Int. J. Pharm. 1999; 178:231-243). Other studies based onanimal testing showed the possibilities of low-MW CS for treatment oftype 2 diabetes and gastric ulcer (Biol. Pharm. Bull. 2002; 25:188-192).Several hydrolytic enzymes such as lysozyme, pectinase, cellulase,bromelain, hemicellulase, lipase, papain and the like can be used todepolymerize CS (Biochim. Biophys. Acta 1996; 1291:5-15; Biochem. Eng.J. 2001; 7:85-88; Carbohydr. Res. 1992; 237:325-332).

FIG. 1 a shows GPC chromatograms of both standard-MW (also known asregular-MW) and low-MW CS. It is known that cellulase catalyzes thecleavage of the glycosidic linkage in CS (Food Chem. 2004; 84:107-115).The low-MW CS used in the study was obtained by precipitating thedepolymerized CS solution with aqueous NaOH at pH 7.0-7.2. Thus,obtained low-MW CS had a MW of about 50 kDa (FIG. 1 a). In a preferredembodiment, the low molecular weight chitosan has a molecular weight ofless than about 40 kDa, but above 10 kDa. Other forms of chitosan mayalso be applicable, including chitin, chitosan oligosaccharides, andderivatives thereof.

It was observed that the obtained low-MW CS can be readily dissolved inan aqueous solution at pH 6.0, while that before depolymerization needsto be dissolved in an acetic acid solution with a pH value about 4.0.Additionally, it was found that with the low-MW CS, the preparednanoparticles had a significantly smaller size with a narrowerdistribution than their counterparts prepared with the high-MW (alsoknown as standard-MW) CS (before depolymerization), due to its lowerviscosity. As an example, upon adding a 0.10% γ-PGA aqueous solutioninto a 0.20% high-MW CS solution (viscosity 5.73±0.08 cp, measured by aviscometer), the mean particle size of the prepared nanoparticles was878.3±28.4 nm with a polydispersity index of 1.0, whereas adding a 0.10%γ-PGA aqueous solution into the low-MW CS solution (viscosity 1.29±0.02cp) formed nanoparticles with a mean particle size of 218.1±4.1 nm witha polydispersity index of 0.3 (n=5).

Example No. 3 Production and Purification of γ-PGA

γ-PGA was produced by Bacillus licheniformis (ATCC 9945, BioresourcesCollection and Research Center, Hsinchu, Taiwan) as per a methodreported by Yoon et al. with slight modifications (Biotechnol. Lett.2000; 22:585-588). Highly mucoid colonies (ATCC 9945a) were selectedfrom Bacillus licheniformis (ATCC 9945) cultured on the E medium(ingredients comprising L-glutamic acid, 20.0 g/l; citric acid, 12.0g/l; glycerol, 80.0 g/l; NH₄Cl, 7.0 g/l; K₂HPO₄, 0.5 g/l; MgSO₄.7H₂O,0.5 g/l; FeCl₃.6H₂O, 0.04 g/l; CaCl₂.2H₂O, 0.15 g/l; MnSO₄.H₂O, 0.104g/l, pH 6.5) agar plates at 37° C. for several times. Subsequently,young mucoid colonies were transferred into 10 ml E medium and grown at37° C. in a shaking incubator at 250 rpm for 24 hours. Afterward, 500 μlof culture broth was mixed with 50 ml E medium and was transferred intoa 2.5-1 jar-fermentor (KMJ-2B, Mituwa Co., Osaka, Japan) containing 950ml of E medium. Cells were cultured at 37° C. The pH was controlled at6.5 by automatic feeding of 25% (v/v) NH₄OH and/or 2M HCl. The dissolvedoxygen concentration was initially controlled at 40% of air saturationby supplying air and by controlling the agitation speed up to 1000 rpm.

After 40 hours, cells were separated from the culture broth bycentrifugation for 20 minutes at 12,000×g at 4° C. The supernatantcontaining γ-PGA was poured into 4 volumes of methanol and leftovernight with gentle stirring. The resulting precipitate containingcrude γ-PGA was collected by centrifugation for 40 minutes at 12,000×gat 4° C. and then was dissolved in deionized water to remove insolubleimpurities by centrifugation for 20 minutes at 24,000×g at 4° C. Theaqueous γ-PGA solution was desalted by dialysis (MWCO: 100,000, SpectrumLaboratories, Inc., Laguna Hills, Calif.) against distilled water for 12hours with water exchanges several times, and finally was lyophilized toobtain pure γ-PGA.

The purified γ-PGA was verified by the proton nuclear magnetic resonance(¹H-NMR) and the FT-IR analyses. Analysis of ¹H-NMR was conducted on anNMR spectrometer (Varian Unityionva 500 NMR Spectrometer, MO) usingDMSO-d₆ at 2.49 ppm as an internal reference. Test samples used for theFT-IR analysis first were dried and ground into a powder form. Thepowder then was mixed with KBr (1:100) and pressed into a disk. Analysiswas performed on an FT-IR spectrometer (Perkin Elmer Spectrum RX1 FT-IRSystem, Buckinghamshire, England). The samples were scanned from400-4000 cm⁻¹. The average molecular weight of the purified γ-PGA wasdetermined by the same GPC system as described before. Polyethyleneglycol (molecular weights of 106-22,000) and polyethylene oxide(molecular weights of 20,000-1,000,000, PL Laboratories) standards wereused to construct a calibration curve. The mobile phase contained 0.01MNaH₂PO₄ and 0.2M NaNO₃ and was brought to a pH of 7.0.

The purified γ-PGA obtained from fermentation was analyzed by GPC,¹H-NMR, and FT-IR. As analyzed by GPC (FIG. 1 b), the purified γ-PGA hada MW of about 160 kDa. In the FT-IR spectrum (FIG. 2 a), acharacteristic peak at 1615 cm⁻¹ for the associated carboxylic acid salt(—COO⁻ antisymmetric stretch) on γ-PGA was observed. The characteristicabsorption due to C═O in secondary amides (amide 1 band) was overlappedby the characteristic peak of —COO⁻. Additionally, the characteristicpeak observed at 3400 cm⁻¹ was the N—H stretch of γ-PGA. In the ¹H-NMRspectrum (FIG. 2 b), six chief signals were observed at 1.73 and 1.94ppm (β-CH₂), 2.19 ppm (γ-CH₂), 4.14 ppm (α-CH), 8.15 ppm (amide), and12.58 ppm (COOH). These results indicated that the observed FT-IR and¹H-NMR spectra correspond well to those expected for γ-PGA.Additionally, the fermented product after purification showed nodetected macromolecular impurities by the ¹H-NMR analysis, suggestingthat the obtained white power of γ-PGA is highly pure.

Example No. 4 Preparation of the CSγ-PGA Nanoparticles

Nanoparticles were obtained upon addition of γ-PGA aqueous solution (pH7.4, 2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®, BrandTechScientific Inc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10ml) at varying concentrations (0.01%, 0.05%, 0.10%, 0.15%, or 0.20% byw/v) under magnetic stirring at room temperature. Nanoparticles werecollected by ultracentrifugation at 38,000 rpm for 1 hour. Supernatantswere discarded and nanoparticles were resuspended in deionized water forfurther studies. FT-IR was used to analyze peak variations of aminogroups of low-MW CS and carboxylic acid salts of 7-PGA in the CSγ-PGAnanoparticles.

As stated, nanoparticles were obtained instantaneously upon addition ofa γ-PGA aqueous solution (pH 7.4) into a low-MW CS aqueous solution (pH6.0) under magnetic stirring at room temperature. FIG. 3 shows the FT-IRspectra of the low-MW CS and the CS-γ-PGA nanoparticles. As shown in thespectrum of CS, the characteristic peak observed at 1563 cm⁻¹ was theprotonated amino group (—NH₃ ⁺ deformation) on CS. In the spectrum ofCS-γ-PGA complex, the characteristic peak at 1615 cm⁻¹ for —COO⁻ onγ-PGA disappeared and a new peak at 1586 cm⁻¹ appeared, while thecharacteristic peak of —NH₃ ⁺ deformation on CS at 1563 cm⁻¹ shifted to1555 cm⁻¹. These observations are attributed to the electrostaticinteraction between the negatively charged carboxylic acid salts (—COO⁻)on γ-PGA and the positively charged amino groups (—NH₃ ⁺) on CS (Int. J.Pharm. 2003; 250:215-226). The electrostatic interaction between the twopolyelectrolytes (γ-PGA and CS) instantaneously induced the formation oflong hydrophobic segments (or at least segments with a high density ofneutral ion-pairs), and thus resulted in highly neutralized complexesthat segregated into colloidal nanoparticles (Langmuir. 2004;20:7766-7778).

Example No. 5 Characterization of the CS-γ-PGA Nanoparticles

The morphological examination of the CS-γ-PGA nanoparticles wasperformed by TEM (transmission electron microscopy) and AFM (atomicforce microscopy). The TEM sample was prepared by placing a drop of thenanoparticle solution onto a 400 mesh copper grid coated with carbon.About 2 minutes after deposition, the grid was tapped with a filterpaper to remove surface water and positively stained by using analkaline bismuth solution (Microbiol. Immunol. 1986; 30:1207-1211). TheAFM sample was prepared by casting a drop of the nanoparticle solutionon a slide glass and then dried in vacuum. The size distribution andzeta potential of the prepared nanoparticles were measured using aZetasizer (3000HS, Malvern Instruments Ltd., Worcestershire, UK).

During storage, aggregation of nanoparticles may occur and thus leads tolosing their structural integrity or forming precipitation ofnanoparticles (Eur. J. Pharm. Sci. 1999; 8:99-107). Therefore, thestability of nanoparticles during storage must be evaluated. In thestability study, the prepared nanoparticles suspended in deionized water(1 mg/ml) were stored at 4° C. and their particle sizes and zetapotential values were monitored by the same Zetasizer as mentionedearlier during storage.

In the preparation of nanoparticles, samples were visually analyzed andthree distinct solution systems were identified: clear solution,opalescent suspension, and solution with precipitation of aggregates.Examined by the Zetasizer, nanoparticles were found in the clearsolution and the opalescent suspension rather than in the solution withprecipitation of aggregates.

The particle sizes and the zeta potential values of CS-γ-PGAnanoparticles, prepared at varying concentrations of γ-PGA and CS, weredetermined and the results are shown in Tables 1a and 1b. It was foundthat the particle size and the zeta potential value of the preparednanoparticles were mainly determined by the relative amount of the localconcentration of γ-PGA in the added solution to the surroundingconcentration of CS in the sink solution. At a fixed concentration ofCS, an increase in the γ-PGA concentration allowed γ-PGA moleculesinteracting with more CS molecules, and thus formed a lager size ofnanoparticles (Table 1a, p<0.05). When the amount of CS moleculesexceeded that of local γ-PGA molecules, some of the excessive CSmolecules were entangled onto the surfaces of CS-γ-PGA nanoparticles.

Thus, the resulting nanoparticles may display a structure of a neutralpolyelectrolyte-complex core surrounded by a positively charged CS shell(Table 1b) ensuring the colloidal stabilization (Langmuir. 2004;20:7766-7778). In contrast, as the amount of local γ-PGA moleculessufficiently exceeded that of surrounding CS molecules, the formednanoparticles had γ-PGA exposed on the surfaces and thus had a negativecharge of zeta potential. Therefore, the particle size and the zetapotential value of the prepared CS-γ-PGA nanoparticles can be controlledby their constituted compositions. The results obtained by the TEM andAFM examinations showed that the morphology of the preparednanoparticles was spherical in shape with a smooth surface (FIGS. 4 aand 4 b). Some aspects of the invention relate to nanoparticles having amean particle size between about 50 and 400 nanometers, preferablybetween about 100 and 300 nanometers, and most preferably between about100 and 200 nanometers. The morphology of the nanoparticles showsspherical in shape with a smooth surface at any pH between 2.5 and 6.6.In one embodiment, the stability of the nanoparticles of the presentinvention at a low pH around 2.5 enables the nanoparticles to be intactwhen exposed to the acidic medium in the stomach.

Two representative groups of the prepared nanoparticles were selectedfor the stability study: one with a positive surface charge (0.10%γ-PGA:0.20% CS) and the other with a negative surface charge (0.10%γ-PGA:0.01% CS). FIG. 5 shows changes in particle size (□, meandiameter) and zeta potential (●) of (a) the CS-γ-PGA nanoparticles(0.10% γ-PGA:0.20% CS) and (b) the CS-γ-PGA nanoparticles (0.10%γ-PGA:0.01% CS) during storage for up to 6 weeks. It was found thatneither aggregation nor precipitation of nanoparticles was observedduring storage for up to 6 weeks, as a result of the electrostaticrepulsion between the

TABLE 1a Effects of concentrations of γ-PGA and CS on the particle sizesof the prepared CS-γ-PGA nanoparticles Mean Particle Size (nm, n = 5) CSγ-PGA 0.01%^(a)) 0.05% 0.10% 0.15% 0.20% 0.01%^(b))  79.0 ± 3.0 103.1 ±4.6  96.7 ± 1.9 103.6 ± 140.5 ± 1.9 2.0 0.05% 157.4 ± 1.7 120.8 ± 3.9144.5 ± 2.4 106.2 ± 165.4 ± 3.8 1.7 0.10% 202.2 ± 3.1 232.6 ± 1.2 161.0± 1.8 143.7 ± 218.1 ± 2.7 4.1 0.15% 277.7 ± 3.2 264.9 ± 2.1 188.6 ± 2.9178.0 ± 301.1 ± 2.2 6.4 0.20% 284.1 ± 2.1 402.2 ± 4.0 ▴ 225.5 ± 365.5 ±3.1 5.1 ^(a))concentration of CS (by w/v) ^(b))concentration of γ-PGA(by w/v) ▴ precipitation of aggregates was observed

TABLE 1b Effects of concentrations of γ-PGA and CS on the zeta potentialvalues of the prepared CS-γ-PGA nanoparticles. Zeta Potential (mV, n =5) CS γ-PGA 0.01%^(a)) 0.05% 0.10% 0.15% 0.20% 0.01%^(b))   15.4 ± 0.322.8 ± 0.5 19.8 ± 1.5 16.5 ± 1.4 17.2 ± 1.6 0.05% −32.7 ± 0.7 23.7 ± 1.727.6 ± 0.7 20.3 ± 0.8 19.2 ± 0.6 0.10% −33.1 ± 1.3 21.1 ± 1.6 20.3 ± 1.123.6 ± 0.9 24.7 ± 1.2 0.15% −33.2 ± 2.1 −21.9 ± 2.0   19.2 ± 0.4 16.9 ±1.7 19.8 ± 0.3 0.20% −34.5 ± 0.5 −34.6 ± 0.3   ▴ 14.6 ± 0.7 16.3 ± 0.7^(a))concentration of CS (by w/v) ^(b))concentration of γ-PGA (by w/v) ▴precipitation of aggregates was observedpositively charged CS-γ-PGA nanoparticles (for the former group) or thenegatively charged CS-γ-PGA nanoparticles (for the latter group).

Additionally, changes in particle size and zeta potential of thenanoparticles were minimal for both studied groups (FIGS. 5 a and 5 b).These results demonstrated that the prepared nanoparticles suspended indeionized water were stable during storage.

In a further study, NPs were self-assembled instantaneously uponaddition of an aqueous γ-PGA into an aqueous TMC/γ-trimethyl chitosan)having a TMC/γ-PGA weight ratio of 6:1 under magnetic stirring at roomtemperature. The chemical formulas of chitosan and N-trimethyl chitosanare shown below:

The amount of positively charged TMC significantly exceeded that ofnegatively charged γ-PGA; some of excessive TMC molecules were entangledonto the surfaces of NPs, thus displaying a positive surface charge(Table 2). The degree of quaternization on TMC had little effects on themean particle size and zeta potential of NPs.

TABLE 2 Mean particle sizes, zeta potential values and polydispersityindices of nanoparticles (NPs) self-assembled by TMC polymers withdifferent degrees of quaternization and γ-PGA (n = 5 batches). MeanParticle Zeta Potential Polydispersity Size (nm) (mV) Index CS/γ-PGA NPs104.1 ± 1.2 36.2 ± 2.5 0.11 ± 0.02 TMC25/γ-PGA NPs 101.3 ± 3.1 30.9 ±2.1 0.13 ± 0.04 TMC40/γ-PGA NPs 106.3 ± 2.3 32.3 ± 2.1 0.15 ± 0.14TMC55/γ-PGA NPs 114.6 ± 2.3 30.6 ± 3.8 0.12 ± 0.03 TMC: N-trimethylchitosan; CS: chitosan; γ-PGA: poly(γ-glutamic acid).

Example No. 6 Caco-2 Cell Cultures and TEER Measurements

Caco-2 cells were seeded on the tissue-culture-treated polycarbonatefilters (diameter 24.5 mm, growth area 4.7 cm²) in Costar Transwell 6wells/plates (Corning Costar Corp., NY) at a seeding density of 3×10⁵cells/insert. MEM (pH 7.4) supplemented with 20% FBS, 1% NEAA, and 40μg/ml antibiotic-gentamicin was used as the culture medium, and added toboth the donor and acceptor compartments. The medium was replaced every48 hours for the first 6 days and every 24 hours thereafter. Thecultures were kept in an atmosphere of 95% air and 5% CO₂ at 37° C. andwere used for the paracellular transport experiments 18-21 days afterseeding (TEER values in the range of 600-800 Ω·cm²).

TEER values of the Caco-2 cell monolayers were monitored with aMillicell®-Electrical Resistance System (Millipore Corp., Bedford,Mass.) connected to a pair of chopstick electrodes. To initiate thetransport experiments, the culture media in the donor and acceptorcompartments were aspirated, and the cells were rinsed twice withpre-warmed transport media (HBSS supplemented with 25 mM glucose, pH6.0). Following a 30-min equilibration with the transport media at 37°C., the cells were incubated for 2 hours with 2 ml transport mediacontaining 0.5 ml test nanoparticle solutions (0.2 mg/ml) at 37° C.Subsequently, solutions of nanoparticles were carefully removed andcells were washed three times with HBSS and replaced by fresh culturemedia. The TEER was measured for another 20 hours to study reversibilityof the effect of test nanoparticles on Caco-2 cell monolayers (Eur. J.Pharm. Sci. 2000; 10:205-214).

The intercellular tight junction is one of the major barriers to theparacellular transport of macromolecules (J. Control. Release 1996;39:131-138; J. Control. Release 1998; 51:35-46). Trans-epithelial iontransport is contemplated to be a good indication of the tightness ofthe junctions between cells and was evaluated by measuring TEER ofCaco-2 cell monolayers in the study. It was reported that themeasurement of TEER can be used to predict the paracellular transport ofhydrophilic molecules (Eur. J. Pharm. Biopharm. 2004; 58:225-235). Whenthe tight junctions open, the TEER value will be reduced due to thewater and ion passage through the paracellular route. Caco-2 cellmonolayers have been widely used as an in vitro model to evaluate theintestinal paracellular permeability of macromolecules.

Effects of the prepared CS-γ-PGA nanoparticles on the TEER values ofCaco-2 cell monolayers are shown in FIG. 6. As shown, the preparednanoparticles with a positive surface charge (CS dominated on thesurface, 0.01% γ-PGA:0.05% CS, 0.10% γ-PGA:0.2% CS, and 0.20%γ-PGA:0.20% CS) were able to reduce the values of TEER of Caco-2 cellmonolayers significantly (p<0.05). After a 2-hour incubation with thesenanoparticles, the TEER values of Caco-2 cell monolayers were reduced toabout 50% of their initial values as compared to the control group(without addition of nanoparticles in the transport media). Thisindicated that the nanoparticles with CS dominated on the surfaces couldeffectively open or loosen the tight junctions between Caco-2 cells,resulting in a decrease in the TEER values. It was reported thatinteraction of the positively charged amino groups of CS with thenegatively charged sites on cell surfaces and tight junctions induces aredistribution of F-actin and the tight junction's protein ZO-1, whichaccompanies the increased paracellular permeability (Drug Deliv. Rev.2001; 50:S91-S101). It is suggested that an interaction between chitosanand the tight junction protein ZO-1, leads to its translocation to thecytoskeleton.

After removal of the incubated nanoparticles, a gradual increase in TEERvalues was noticed. This phenomenon indicated that the intercellulartight junctions of Caco-2 cell monolayers started to recover gradually;however, the TEER values did not recover to their initial values (FIG.6). Kotze et al. reported that complete removal of a CS-derived polymer,without damaging the cultured cells, was difficult due to the highlyadhesive feature of CS (Pharm. Res. 1997; 14:1197-1202). This might bethe reason why the TEER values did not recover to their initial values.In contrast, the TEER values of Caco-2 cell monolayers incubated withthe nanoparticles with a negative surface charge (γ-PGA dominated on thesurface, 0.10% γ-PGA:0.01% CS and 0.20% γ-PGA:0.01% CS, FIG. 6) showedno significant differences as compared to the control group (p>0.05).This indicated that γ-PGA does not have any effects on the opening ofthe intercellular tight junctions.

FIG. 8 shows an illustrative protein transport mechanism through acellular layer, including transcellular transport and paracellulertransport. FIG. 9 shows a schematic illustration of a paracellulartransport mechanism. The transcellular protein or peptide transport maybe an active transport or a passive transport mode whereas theparacellular transport is basically a passive mode. Ward et al. reportedand reviewed current knowledge regarding the physiological regulation oftight junctions and paracellular permeability (PSTT 2000; 3:346-358).Chitosan as nanoparticle vehicles for oral delivery of protein drugsavoids the enzymatic inactivation in the gastrointestinal conduit. Thechitosan component of the present nanoparticles has a special feature ofadhering to the mucosal surface and transiently opening the tightjunctions between epithelial cells; that is, loosening the tightness ofthe tight junctions.

FIG. 9(A) shows that after feeding nanoparticles (NPs) orally, NPsadhere and infiltrate into the mucus layer of the epithelial cells. FIG.9(B) illustrates that the infiltrated NPs transiently and reversiblyloosen tight junctions (TJs) while becoming unstable and disintegratedto release insulin or entrapped agent. FIG. 9( c) shows that thereleased insulin or agent permeates through the paracellular pathwayinto the blood stream. Chitosan (CS), a nontoxic, soft-tissuecompatible, cationic polysaccharide has special features of adhering tothe mucosal surface; CS is able to transiently and reversiblywiden/loosen TJs between epithelial cells. The TJ width in the smallintestine has been demonstrated to be less than 1 nm. It is also knownthat TJs ‘opened’ by absorption enhancers are less than 20 nm wide(Nanotechnology 2007; 18:1-11). The term “opened” herein means that anysubstance less than 20 nm in the close-proximity might have the chanceto pass through. TJs constitute the principal barrier to passivemovement of fluid, electrolytes, macromolecules and cells through theparacellular pathway.

It was suggested that the electrostatic interaction between thepositively charged CS and the negatively charged sites of ZO-1 proteinson cell surfaces at TJ induces a redistribution of cellular F-actin andZO-1's translocation to the cytoskeleton, leading to an increase inparacellular permeability. As evidenced in FIG. 9, after adhering andinfiltrating into the mucus layer of the duodenum, the orallyadministered nanoparticles may degrade due to the presence of distinctdigestive enzymes in the intestinal fluids. Additionally, the pHenvironment may become neutral while the nanoparticles were infiltratinginto the mucosa layer and approaching the intestinal epithelial cells.This further leads to the collapse of nanoparticles due to the change inthe exposed pH environment. The dissociated CS from thedegraded/collapsed nanoparticles was then able to interact and modulatethe function of ZO-1 proteins between epithelial cells (Nanotechnology2007; 18:1-11). ZO-1 proteins are thought to be a linkage moleculebetween occludin and F-actin cytoskeleton and play important roles inthe rearrangement of cell-cell contacts at TJs.

Example No. 7 fCS-γ-PGA Nanoparticle Preparation and CLSM Visualization

Fluorescence (FITC)-labeled CS-γ-PGA (fCS-γ-PGA) nanoparticles (FIG. 10)were prepared for the confocal laser scanning microscopy (CLSM) study.The nanoparticles of the present invention display a structure of aneutral polyelectrolyte-complex core surrounded by a positively chargedchitosan shell. Synthesis of the FITC-labeled low-MW CS (fCS) was basedon the reaction between the isothiocyanate group of FITC and the primaryamino groups of CS as reported in the literature (Pharm. Res. 2003;20:1812-1819). Briefly, 100 mg of FITC in 150 ml of dehydrated methanolwere added to 100 ml of 1% low-MW CS in 0.1M acetic acid. After 3 hoursof reaction in the dark at ambient conditions, fCS was precipitated byraising the pH to about 8-9 with 0.5M NaOH. To remove the unconjugatedFITC, the precipitate was subjected to repeated cycles of washing andcentrifugation (40,000×g for 10 min) until no fluorescence was detectedin the supernatant. The fCS dissolved in 80 ml of 0.1M acetic acid wasthen dialyzed for 3 days in the dark against 5 liters of distilledwater, with water replaced on a daily basis. The resultant fCS waslyophilized in a freeze dryer. The fCS-γ-PGA nanoparticles were preparedas per the procedure described in Example No. 4.

Subsequently, the transport medium containing fCS-γ-PGA nanoparticles(0.2 mg/ml) was introduced into the donor compartment of Caco-2 cells,which were pre-cultured on the transwell for 18-21 days. Theexperimental temperature was maintained at 37° C. by a temperaturecontrol system (DH-35 Culture Dish Heater, Warner Instruments Inc.,Hamden, Conn.). After incubation for specific time intervals, testsamples were aspirated. The cells were then washed twice with pre-warmedPBS solution before they were fixed in 3.7% paraformaldehyde (Pharm.Res. 2003; 20:1812-1819). Cells were examined under an inversed CLSM(TCS SL, Leica, Germany). The fluorescence images were observed using anargon laser (excitation at 488 nm, emission collected at a range of510-540 nm).

CLSM was used to visualize the transport of the fluorescence-labeledCS-γ-PGA (fCS-γ-PGA) nanoparticles across the Caco-2 cell monolayers.This non-invasive method allows for optical sectioning and imaging ofthe transport pathways across the Caco-2 cell monolayers, withoutdisrupting their structures (J. Control. Release 1996; 39:131-138).FIGS. 7 a and 7 b show the fluorescence images of 4 optical sections ofa Caco-2 cell monolayer that had been incubated with the fCS-γ-PGAnanoparticles having a positive surface charge (0.10% γ-PGA:0.20% CS,zeta potential: about 21 mV) for 20 and 60 min, respectively. As shown,after 20 min of incubation with the nanoparticles, intense fluorescencesignals at intercellular spaces were observed at depths of 0 and 5 μmfrom the apical (upper) surface of the cell monolayer. The intensity offluorescence became weaker at levels deeper than 10 μm from the apicalsurface of the cell monolayer and was almost absent at depths ≧15 μm(FIG. 7 a).

After 60 minutes of incubation with the nanoparticles, the intensity offluorescence observed at intercellular spaces was stronger and appearedat a deeper level than those observed at 20 min after incubation. Theseobservations confirmed with our TEER results that the nanoparticles witha positive surface charge (CS dominated on the surface) were able toopen the tight junctions between Caco-2 cells and allowed transport ofthe nanoparticles by passive diffusion via the paracellular pathways.

Example No. 8 In Vivo Study with Fluorescence-labeled Nanoparticles

Fluorescence (FITC)-labeled CS-γ-PGA (fCS-γ-PGA) nanoparticles wereprepared for the confocal laser scanning microscopy (CLSM) study. Afterfeeding rats with fCS-γ-PGA nanoparticles, the rats are sacrificed at apre-determined time and the intestine is isolated for CLSM examination.The fluorescence images of the nanoparticles were clearly observed byCLSM that penetrates through the mouse intestine at appropriate time andat various depths from the inner surface toward the exterior surface ofthe intestine, including duodenum, jejunum, and ileum.

Example No. 9 Insulin Loading Capacity in Nanoparticles

Fluorescence (FITC)-labeled γ-PGA was added into chitosan solution toprepare fluorescence (FITC)-labeled, insulin-loaded CS-γ-PGAnanoparticles for in vivo animal study with confocal laser scanningmicroscopy (CLSM) assessment and bioactivity analysis. Theinsulin-loaded CS-γPGA nanoparticles are by using the ionic-gelationmethod upon addition of insulin mixed with γ-PGA solution into CSsolution, followed by magnetic stirring in a container.

Model insulin used in the experiment and disclosed herein is obtainedfrom bovine pancreas (Sigma-Aldrich, St. Louis, Mo.), having a molecularformula of C₂₅₄H₃₇₇N₆₅O₇₅S₆ with a molecular weight of about 5733.5 andan activity of ≧27 USP units/mg. The insulin contains two-chainpolypeptide hormone produced by the β-cells of pancreatic islets. The αand βchains are joined by two interchain disulfide bonds. Insulinregulates the cellular uptake, utilization, and storage of glucose,amino acids, and fatty acids and inhibits the breakdown of glycogen,protein, and fat. The insulin from Sigma-Aldrich contains about 0.5%zinc. Separately, insulin can be obtained from other sources, such ashuman insulin solution that is chemically defined, recombinant fromSaccharomyces cerevisiae. Some aspects of the invention relate tonanoparticles with insulin in the core, wherein the insulin may containintermediate-acting, regular insulin, rapid-acting insulin,sustained-acting insulin that provides slower onset and longer durationof activity than regular insulin, or combinations thereof.

Examples of insulin or insulin analog products include, but not limitedto, Humulin® (by Eli Lilly), Humalog® (by Eli Lilly) and Lantus® (byAventis), and Novolog®& Mix70/30 (by Novo Nordisk). Humalog (insulinlispro, rDNA origin) is a human insulin analog that is a rapid-acting,parenteral blood glucose-lowering agent. Chemically, it is Lys(B28),Pro(B29) human insulin analog, created when the amino acids at positions28 and 29 on the insulin B-chain are reversed. Humalog is synthesized ina special non-pathogenic laboratory strain of Escherichia coli bacteriathat has been genetically altered by the addition of the gene forinsulin lispro. Humalog has the empirical formula C₂₅₇H₃₈₃N₆₅O₇₇S₆ and amolecular weight of 5808, identical to that of human insulin. The vialsand cartridges contain a sterile solution of Humalog for use as aninjection. Humalog injection consists of zinc-insulin lispro crystalsdissolved in a clear aqueous fluid. Each milliliter of Humalog injectioncontains insulin lispro 100 Units, 16 mg glycerin, 1.88 mg dibasicsodium phosphate, 3.15 mg m-cresol, zinc oxide content adjusted toprovide 0.0197 mg zinc ion, trace amounts of phenol, and water forinjection. Insulin lispro has a pH of 7.0-7.8. Hydrochloric acid 10%and/or sodium hydroxide 10% may be added to adjust pH.

Humulin is used by more than 4 million people with diabetes around theworld every day. Despite its name, this insulin does not come from humanbeings. It is identical in chemical structure to human insulin and ismade in a factory using a chemical process called recombinant DNAtechnology. Humulin L is an amorphous and crystalline suspension ofhuman insulin with a slower onset and a longer duration of activity (upto 24 hours) than regular insulin. Humulin U is a crystalline suspensionof human insulin with zinc providing a slower onset and a longer andless intense duration of activity (up to 28 hours) than regular insulinor the intermediate-acting insulins (NPH and Lente).

LANTUS® (insulin glargine [rDNA origin] injection) is a sterile solutionof insulin glargine for use as an injection. Insulin glargine is arecombinant human insulin analog that is a long-acting (up to 24-hourduration of action), parenteral blood-glucose-lowering agent. LANTUS isproduced by recombinant DNA technology utilizing a non-pathogeniclaboratory strain of Escherichia coli (K12) as the production organism.Insulin glargine differs from human insulin in that the amino acidasparagine at position A21 is replaced by glycine and two arginines areadded to the C-terminus of the B-chain. Chemically, it is21^(A)-Gly-30^(B)a-L-Arg-30^(B)b-L-Arg-human insulin and has theempirical formula C₂₆₇H₄₀₄N₇₂O₇₈S₆ and a molecular weight of 6063.

LANTUS consists of insulin glargine dissolved in a clear aqueous fluid.Each milliliter of LANTUS (insulin glargine injection) contains 100 IU(3.6378 mg) insulin glargine. Inactive ingredients for the 10 mL vialare 30 mcg zinc, 2.7 mg m-cresol, 20 mg glycerol 85%, 20 mcg polysorbate20, and water for injection. Inactive ingredients for the 3 mL cartridgeare 30 mcg zinc, 2.7 mg m-cresol, 20 mg glycerol 85%, and water forinjection. In 2006, there were 11.4 million prescriptions of Lantus inthe U.S. for basal insulin maintenance.

Novolog® Mix70/30 (70% insulin aspart protamine suspension and 30%insulin aspart injection [rDNA origin]) is a human insulin analogsuspension. Novolog® Mix70/30 is a blood glucose-lowering agent with arapid onset and an intermediate duration of action. Insulin aspart ishomologous with regular human insulin with the exception of a singlesubstitution of the amino acid praline by aspartic acid in position B28,and is produced by recombinant DNA technology utilizing Saccharomycescerevisiae as the production organism. Insulin aspart (Novolog) has theempirical formula C₂₅₆H₃₈₁N₆₅O₇₉S₆ and a molecular weight of 5826.Novolog® Mix70/30 is a uniform, white sterile suspension that containszinc 19.6 μg/ml and other components.

The nanoparticles with two insulin concentrations are prepared at achitosan to γ-PGA ratio of 0.75 mg/ml to 0.167 mg/ml. Their particlesize and zeta potential are shown in Table 3 below.

TABLE 3 Polydispersity Zeta Insulin Conc. Mean Particle Index Potential(mg/ml) (n = 5) Size (nm) (PI) (mV) 0* 145.6 ± 1.9 0.14 ± 0.01 +32.11 ±1.61 0.042 185.1 ± 5.6 0.31 ± 0.05 +29.91 ± 1.02 0.083 198.4 ± 6.2 0.30± 0.09 +27.83 ± 1.22 *control reference without insulin

Further, their association efficiency of insulin and loading capacity ofinsulin are analyzed, calculated and shown in FIGS. 11 and 12, accordingto the following formula:

${{Insulin}\mspace{14mu}{Association}} = {\frac{\begin{pmatrix}{{{Total}\mspace{14mu}{amount}\mspace{14mu}{of}\mspace{14mu}{insulin}} -} \\{{Insulin}\mspace{14mu}{in}\mspace{14mu}{supernatant}}\end{pmatrix}}{{Total}\mspace{14mu}{amount}\mspace{14mu}{of}\mspace{14mu}{insulin}} \times 100\%}$${{Efficiency}\mspace{14mu}\left( {{LE}\mspace{14mu}\%} \right)\mspace{14mu}{Loading}\mspace{14mu}{Capacity}\mspace{14mu}\left( {L\; C} \right)} = {\frac{\begin{pmatrix}{{{Total}\mspace{14mu}{amount}\mspace{14mu}{of}\mspace{14mu}{insulin}} -} \\{{Insulin}\mspace{14mu}{in}\mspace{14mu}{supernatant}}\end{pmatrix}}{{Weight}\mspace{14mu}{of}\mspace{14mu}{recovered}\mspace{14mu}{particles}} \times 100\%}$

FIG. 11 shows loading capacity and association efficiency of insulin innanoparticles of chitosan and γ-PGA, whereas FIG. 12 shows loadingcapacity and association efficiency of insulin in nanoparticles ofchitosan alone (in absence of γ-PGA) as reference. The data clearlydemonstrates that both the insulin loading capacity and insulinassociation efficiency are statistically higher for the nanoparticleswith γ-PGA in the core. The LE (40˜55%) and LC (5.0˜14.0%) of insulinfor CS-γ PGA nanoparticles was obtained by using ionic-gelation methodupon addition of insulin mixed with γ-PGA solution into CS solution,followed by magnetic stirring for nanoparticle separation. In certainfollow-up experiments, nanoparticles having a pharmaceutical compositionhave been successfully prepared with a negatively charged componentcomprised of γ-PGA, α-PGA, PGA derivatives, salts of PGA, heparin orheparin analog, or alginate. The PGA derivatives of the presentinvention may include, but not limited to, poly-γ-glutamic acid,poly-α-glutamic acid, poly-L-glutamic acid (manufactured bySigma-Aldrich, St. Louis, Mo.), poly-D-glutamic acid, poly-L-α-glutamicacid, poly-γ-D-glutamic acid, poly-γ-DL-glutamic acid, and PEG or PHEGderivatives of polyglutamic acid, salts of the above-cited PGAs, and thelike. Some aspects of the invention relate to nanoparticles comprising ashell component and a core component, wherein at least a portion of theshell component comprises chitosan and wherein the core component iscomprised of a negatively charged compound that is conjugated tochitosan, and a bioactive agent. Some aspects of the invention relate toan oral dose of nanoparticles that effectively enhance intestinal orblood brain paracellular transport comprising a negative component (suchas γ-PGA, α-PGA, PGA derivatives, heparin, or alginate) in the core andlow molecular weight chitosan, wherein the chitosan dominates on asurface of the nanoparticles with positive charges.

Some aspects of the invention relate to a dose of nanoparticles thateffectively enhance intestinal or blood brain paracellular transportcomprising a polyanionic component (such as γ-PGA, α-PGA, PGAderivatives, heparin, heparin analogs, low molecular weight heparin,glycosaminoglycans, or alginate) in the core and low molecular weightchitosan in the shell, wherein the chitosan dominates on a surface ofthe nanoparticles with surface positive charges. In use, firstly,encapsulate the Alzheimer's drug in the chitosan shell nanoparticle asdescribed herein, wherein the nanoparticle is partially crosslinked(optionally) to enhance its biodurability. Then intra-venously injectthe nanoparticles, whereby the nanoparticles pass to the brain in bloodcirculation. The chitosan shell of the nanoparticles adheres to thesurface adjacent the tight junction in the brain. Thereafter, thechitosan nanoparticle opens the tight junction, wherein the Alzheimer'sdrug is released after passing the tight junction for therapeutictreatment. In one embodiment, the nanoparticles are in a spherical shapehaving a mean particle size of about 50 to 250 nanometers, preferably150 nanometers to 250 nanometers.

In one example, intravenous administration of the nanoparticlescomprising chitosan shell substrate, polyanionic core substrate and atleast one bioactive agent for treating Alzheimer's disease in a patientis typically performed with 10 mg to 40 mg of active agent per day overa period of one month to one year. The bioactive agent is selected fromthe group consisting of donepezile, rivastignine, galantamine, and/orthose trade-named products, such as memantine hydrochloride (Axura® byMerz Pharmaceuticals), donepezil hydrochloride (Aricept® by Eisai Co.Ltd.), rivastigmine tartrate (Exelon® by Novartis), galantaminehydrochloride (Reminyl® by Johnson & Johnson), and tacrine hydrochloride(Cognex® by Parke Davis).

Some aspects of the invention relate to a nanoparticle with a coresubstrate comprising polyglutamic acids such as water soluble salt ofpolyglutamic acids (for example, ammonium salt) or metal salts ofpolyglutamic acid (for example, lithium salt, sodium salt, potassiumsalt, magnesium salt, and the like). In one embodiment, the form ofpolyglutamic acid may be selected from the group consisting ofpoly-α-glutamic acid, poly-L-α-glutamic acid, poly-γ-glutamic acid,poly-D-glutamic acid, poly-γ-D-glutamic acid, poly-γ-DL-glutamic acid,poly-L-glutamic acid (manufactured by Sigma-Aldrich, St. Louis, Mo.),and PEG or PHEG derivatives of polyglutamic acid. Alginate is generallynon-biodegradable; however, it is stipulated that an alginate particlewith about 30-50 kDa molecular weight is kidney inert. Heparin withnegatively charged side-groups has a general chemical structure as shownbelow:

Some aspects of the invention relate to the negatively chargedglycosaminoglycans (GAGs) as the core substrate of the presentnanoparticles. GAGs may be used to complex with a low-molecular-weightchitosan to form drug-carrier nanoparticles. GAGs may also conjugatewith the protein drugs as disclosed herein to enhance the bondingefficiency of the core substrate in the nanoparticles. Particularly, thenegatively charged core substrate (such as GAGs, heparin, PGA, alginate,and the like) of the nanoparticles of the present invention mayconjugate with chondroitin sulfate, hyaluronic acid, PDGF-BB, BSA, EGF,MK, VEGF, KGF, bFGF, aFGF, MK, PTN, etc.

Calceti et al. reported an in vivo evaluation of an oral insulin-PEGdelivery system (Eur J Pharma Sci 2004; 22:315-323). Insulin-PEG wasformulated into mucoadhesive tablets constituted by the thiolatedpolymer poly(acrylic acid)-cysteine. The therapeutic agent was sustainedreleased from these tablets within 5 hours. In vivo, by oraladministration to diabetic mice, the glucose levels were found todecrease significantly over the time. Further, Krauland et al. reportedanother oral insulin delivery study of thiolated chitosan-insulintablets on non-diabetic rats (J. Control. Release 2004, 95:547-555). Thedelivery tablets utilized 2-Iminothiolane covalently linked to chitosanto form chitosan-TBA (chitosan-4-thiobutylamidine) conjugate. After oraladministration of chitosan-TBA-insulin tablets to non-diabetic consciousrats, the blood glucose level decreased significantly for 24 hours;supporting the observation of sustained insulin release of the presentlydisclosed nanoparticles herein through intestinal absorption. In afurther report by Morcol et al. (Int. J. Pharm. 2004; 277:91-97), anoral delivery system comprising calcium phosphate-PEG-insulin-caseinparticles displays a prolonged hypoglycemic effect after oraladministration to diabetic rats.

Pan et al. disclosed chitosan nanoparticles improving the intestinalabsorption of insulin in vivo (Int J Pharma 2002; 249:139-147) withinsulin-chitosan nanoparticles at a particle size of 250-400 nm, apolydispersity index smaller than 0.1, positively charged and stable.After administering the insulin-chitosan nanoparticles, it was foundthat the hypoglycemic was prolonged with enhanced pharmacologicalbioavailability. Their data confirmed our observation as shown in FIGS.11 and 12; however, the insulin loading capacity and insulin associationefficiency of the present invention are substantially higher for thechitosan-insulin nanoparticles with γ-PGA in the core as the coresubstrate.

Example No. 10 Insulin Nanoparticle Stability

FIG. 13 shows the stability of insulin-loaded nanoparticles of thepresent invention with an exemplary composition of CS 0.75 mg/ml, γ-PGA0.167 mg/ml, and insulin 0.083 mg/ml. The prepared insulin-loadednanoparticles suspended in deionized water are stable during storage upto 40 days. First (in FIG. 13), the insulin content in the nanoparticlestorage solution maintains at about a constant level of 9.5%. Thenanoparticle stability is further evidenced by the substantiallyconstant particle size at about 200 nm and substantially constant zetapotential of about +28 mV over the period of about 40 days. It iscontemplated that the insulin-containing nanoparticles of the presentinvention would further maintain their biostability when formulated in asoft gelcap or capsule configuration that further isolates thenanoparticles from environmental effects, such as sunlight, heat, airconditions, and the like. Some aspects of the invention provide a gelcappill or capsule containing a dosage of insulin nanoparticles effectiveamount of the insulin to treat or manage the diabetic patients, whereinthe stability of the insulin-containing nanoparticles is at least 40days, preferably more than 6 months, and most preferably more than acouple of years.

By “effective amount of the insulin”, it is meant that a sufficientamount of insulin will be present in the dose to provide for a desiredtherapeutic, prophylatic, or other biological effect when thecompositions are administered to a host in the single dosage forms. Thecapsule of the present invention may preferably comprise two-parttelescoping gelatin capsules. Basically, the capsules are made in twoparts by dipping metal rods in molten gelatin solution. The capsules aresupplied as closed units to the pharmaceutical manufacturer. Before use,the two halves are separated, the capsule is filled with powder (eitherby placing a compressed slug of powder into one half of the capsule, orby filling one half of the capsule with loose powder) and the other halfof the capsule is pressed on. The advantage of inserting a slug ofcompressed powder is that control of weight variation is better. Thecapsules may be enterically coated before filling the powder or afterfilling the powder and securing both parts together.

Thus, for convenient and effective oral administration, pharmaceuticallyeffective amounts of the nanoparticles of this invention can betabletted with one or more excipient, encased in capsules such as gelcapsules, and suspended in a liquid solution and the like. Thenanoparticles can be suspended in a deionized solution or the like forparenteral administration. The nanoparticles may be formed into a packedmass for ingestion by conventional techniques. For instance, thenanoparticles may be encapsulated as a “hard-filled capsule” or a“soft-elastic capsule” using known encapsulating procedures andmaterials. The encapsulating material should be highly soluble ingastric fluid so that the particles are rapidly dispersed in the stomachafter the capsule is ingested. Each unit dose, whether capsule ortablet, will preferably contain nanoparticles of a suitable size andquantity that provides pharmaceutically effective amounts of thenanoparticles. The applicable shapes and sizes of capsules may includeround, oval, oblong, tube or suppository shape with sizes from 0.75 mmto 80 mm or larger. The volume of the capsules can be from 0.05 cc tomore than 5 cc.

Example No. 11 In Vitro Insulin Release Study

FIG. 14 show a representative protein drug (for example, insulin)release profile in a pH-adjusted solution for pH-sensitivity study withan exemplary composition of CS 0.75 mg/ml, γ-PGA 0.167 mg/ml, andinsulin 0.083 mg/ml in nanoparticles. In one embodiment, the exemplarycomposition may include each component at a concentration range of ±10%as follows: CS 0.75 mg/ml (a concentration range of 0.67 to 0.83 mg/ml),γ-PGA 0.167 mg/ml (a concentration range of 0.150 to 0.184 mg/ml), andinsulin 0.083 mg/ml (a concentration range of 0.075 to 0.091 mg/ml).First, solution of the insulin-loaded nanoparticles was adjusted to pH2.5 to simulate the gastric environment in a DISTEK-2230A container at37° C. and 100 rpm. Samples (n=5) were taken at a pre-determinedparticular time interval and the particle-free solution was obtained bycentrifuging at 22,000 rpm for 30 minutes to analyze the free orreleased insulin in solution by HPLC. Until the free insulin content inthe sample solution approaches about constant of 26% (shown in FIG. 14),the pH was adjusted to 6.6 to simulate the entrance portion of theintestine. The net released insulin during this particular time intervalis about (from 26% to 33%) 7%. In other words, the nanoparticles arequite stable (evidenced by minimal measurable insulin in solution) forboth the pH 2.5 and pH 6.6 regions. To further simulate the exit portionof the intestine, the insulin-containing nanoparticle solution isadjusted to pH 7.4. The remaining insulin (about 67%) is released fromthe nanoparticles. As discussed above, the insulin in nanoparticleswould be more effective to penetrate the intestine wall in paracellulartransport mode than the free insulin because of the nanoparticles of thepresent invention with chitosan at the outer surface (preferentialmucosal adhesion on the intestinal wall) and positive charge (enhancingparacellular tight junction transport).

Example No. 12 In Vivo Study with Insulin-loaded Fluorescence-labeledNanoparticles

In the in vivo study, rats were injected with streptozotocin (STZ 75mg/kg intraperitoneal) in 0.01M citrate buffer (pH 4.3) to inducediabetes rats. The blood from the rat's tail was analyzed with acommercially available glucometer for blood glucose. The blood glucoselevel on Wistar male rats at no fasting (n=5) is measured at 107.2±8.1mg/dL for normal rats while the blood glucose level is at 469.7±34.2mg/dL for diabetic rats. In the animal study, diabetic rats were fastingfor 12 hours and subjected to four different conditions: (a) oraldeionized water (DI) administration; (b) oral insulin administration at30 U/kg; (c) oral insulin-loaded nanoparticles administration at 30U/kg; and (d) subcutaneous (SC) insulin injection at 5 U/kg as positivecontrol. The blood glucose concentration from rat's tail was measuredover the time in the study.

FIG. 15 shows glucose change (hypoglycemic index) versus time of the invivo animal study (n=5). The glucose change as a percentage of baselines for both oral DI administration and oral insulin administrationover a time interval of 8 hours appears relatively constant within theexperimental measurement error range. It is illustrative thatsubstantially all insulin from the oral administration route has beendecomposed in rat stomach. As anticipated, the glucose decrease for theSC insulin injection route appears in rat blood in the very early timeinterval and starts to taper off after 3 hours in this exemplary study.The most important observation of the study comes from the oraladministration route with insulin-loaded nanoparticles. The bloodglucose begins to decrease from the base line at about 2 hours afteradministration and sustains at a lower glucose level at more than 8hours into study. It implies that the current insulin-loadednanoparticles may modulate the glucose level in animals in a sustainedor prolonged effective mode. Some aspects of the invention provide amethod of treating diabetes of a patient comprising orally administeringinsulin-containing nanoparticles with a dosage effective amount of theinsulin to treat the diabetes, wherein at least a portion of thenanoparticles comprises a positively charged shell substrate and anegatively charged core substrate. In one embodiment, the dosageeffective amount of the insulin to treat the diabetes comprises aninsulin amount of between about 15 units to 45 units per kilogram bodyweight of the patient, preferably 20 to 40 units, and most preferably atabout 25 to 35 units insulin per kilogram body weight.

Some aspects of the invention relate to a novel nanoparticle system thatis composed of a low-MW CS and γ-PGA with CS dominated on the surfacesbeing configured to effectively open the tight junctions between Caco-2cell monolayers. The surface of the nanoparticles is characterized witha positive surface charge. In one embodiment, the nanoparticles of theinvention enables effective intestinal delivery for bioactive agent,including peptide, polypeptide, protein drugs, other large hydrophilicmolecules, and the like. Such polypeptide drugs can be any natural orsynthetic polypeptide that may be orally administered to a humanpatient.

Exemplary drugs include, but are not limited to, insulin; growthfactors, such as epidermal growth factor (EGF), insulin-like growthfactor (IGF), transforming growth factor (TGF), nerve growth factor(NGF), platelet-derived growth factor (PDGF), bone morphogenic protein(BMP), fibroblast growth factor and the like; somatostatin;somatotropin; somatropin; somatrem; calcitonin; parathyroid hormone;colony stimulating factors (CSF); clotting factors; tumor necrosisfactors: interferons; interleukins; gastrointestinal peptides, such asvasoactive intestinal peptide (VIP), cholecytokinin (CCK), gastrin,secretin, and the like; erythropoietins; growth hormone and GRF;vasopressins; octreotide; pancreatic enzymes; dismutases such assuperoxide dismutase; thyrotropin releasing hormone (TRH); thyroidstimulating hormone; luteinizing hormone; LHRH; GHRH; tissue plasminogenactivators; macrophage activator; chorionic gonadotropin; heparin;atrial natriuretic peptide; hemoglobin; retroviral vectors; relaxin;cyclosporin; oxytocin; vaccines; monoclonal antibodies; and the like;and analogs and derivatives of these compounds. The bioactive agent ofthe present invention may be selected from group consisting of oxytocin,vasopressin, adrenocorticotrophic hormone, prolactin, luliberin orluteinising hormone releasing hormone, growth hormone, growth hormonereleasing factor, somatostatin, glucagon, interferon, gastrin,tetragastrin, pentagastrin, urogastroine, secretin, calcitonin,enkephalins, endorphins, angiotensins, renin, bradykinin, bacitracins,polymixins, colistins, tyrocidin, gramicidines, and synthetic analogues,modifications and pharmacologically active fragments thereof, monoclonalantibodies and soluble vaccines.

In another embodiment, the nanoparticles of the invention increase theabsorption of bioactive agents across the blood brain barrier and/or thegastrointestinal barrier. In still another embodiment, the nanoparticleswith chitosan at an outer layer and surface positive charge serve as anenhancer in enhancing paracellular drug (bioactive agent) transport ofan administered bioactive agent when the bioactive agent andnanoparticles are orally administrated in a two-component system, ororally administered substantially simultaneously.

Example No. 13 Paracellular Transport and Enhancers

Chitosan and its derivatives may function as intestinal absorptionenhancers (that is, paracellular transport enhancers). Chitosan, whenprotonated at an acidic pH, is able to increase the paracellularpermeability of peptide drugs across mucosal epithelia. Some aspects ofthe invention provide co-administration of nanoparticles of the presentinvention and at least one paracellular transport enhancer (innon-nanoparticle form or nanoparticle form). In one embodiment, thenanoparticles can be formulated by co-encapsulation of the at least oneparacellular transport enhancer and at least one bioactive agent,optionally with other components. The enhancer may be selected from thegroup consisting of Ca²⁺ chelators, bile salts, anionic surfactants,medium-chain fatty acids, phosphate esters, and chitosan or chitosanderivatives. In one embodiment, the nanoparticles of the presentinvention comprises a positively charged shell substrate and anegatively charged core substrate, for example, nanoparticles composedof γ-PGA and chitosan that is characterized with a substantiallypositive surface charge.

In some embodiment, the nanoparticles of the present invention and theat least one paracellular transport enhancer are encapsulated in a softgel, pill, or enteric coated capsule. The enhancers and thenanoparticles would arrive at the tight junction about the same time forenhancing opening the tight junction. In another embodiment, the atleast one paracellular transport enhancer is co-enclosed within theshell of the nanoparticles of the present invention. Therefore, somebroken nanoparticles would release enhancers to assist the nanoparticlesto open the tight junctions of the epithelial layers. In an alternateembodiment, the at least one enhancer is enclosed within a secondnanoparticle having positive surface charges, particularly a chitosantype nanoparticle. When the drug-containing first nanoparticles of thepresent invention is co-administered with the above-identified secondnanoparticles orally, the enhancers within the second nanoparticles arereleased in the intestinal tract to assist the drug-containing firstnanoparticles to open and pass the tight junction.

Example No. 14 Nanoparticles with Complexed Calcitonin

Calcitonin is a protein drug that serves therapeutically as calciumregulators for treating osteoporosis (J. Pharm. Pharmacol. 1994;46:547-552). Calcitonin has a molecular formula of C₁₄₅H₂₄₀N₄₄O₄₈S₂ witha molecular weight of about 3431.9 and an isoelectric point of 8.7. Thenet charge for calcitonin at pH7.4 is positive that is suitable tocomplex or conjugate with negatively charged core substrate, such asγ-PGA or A-PGA. In preparation, nanoparticles were obtained uponaddition of a mixture of δ-PGA plus calcitonin aqueous solution (pH 7.4,2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®, BrandTech ScientificInc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10 ml) atconcentrations higher than 0.10% by w/v under magnetic stirring at roomtemperature to ensure positive surface charge. Nanoparticles werecollected by ultracentrifugation at 38,000 rpm for 1 hour. Supernatantswere discarded and nanoparticles were resuspended in deionized water asthe solution products, further encapsulated in capsules or furthertreated with an enteric coating.

Example No. 15 Nanoparticles with Conjugated Vancomycin

Vancomycin is a protein drug that serves therapeutically as antibioticagainst bacterial pathogens. Vancomycin has a molecular formula ofC₆₆H₇₅N₉O₂₄ with a molecular weight of about 1485.7 and an isoelectricpoint of 5.0. The net charge for vancomycin at pH7.4 is negative that issuitable to complex or conjugate with a portion of negatively chargedshell substrate, such as chitosan. In preparation, nanoparticles wereobtained upon addition of a mixture of γ-PGA plus vancomycin aqueoussolution (pH 7.4, 2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®,BrandTech Scientific Inc., Germany), into a low-MW CS aqueous solution(pH 6.0, 10 ml) with excess concentrations under magnetic stirring atroom temperature, wherein CS concentration is provided sufficiently toconjugate vancomycin, to counterbalance γ-PGA, and exhibit positivesurface charge for the nanoparticles. Nanoparticles were collected byultracentrifugation at 38,000 rpm for 1 hour. Supernatants werediscarded and nanoparticles were resuspended in deionized water as thesolution products, further encapsulated in capsules or further treatedwith an enteric coating on capsules.

Some aspects of the invention relate to a method of enhancing intestinalor blood brain paracellular transport of bioactive agents configured andadapted for delivering at least one bioactive agent in a patientcomprising administering nanoparticles composed of γ-PGA and chitosan,wherein the nanoparticles are loaded with a therapeutically effectiveamount or dose of the at least one bioactive agent. The nanoparticle ofthe present invention is an effective intestinal delivery system forpeptide and protein drugs and other large hydrophilic molecules. In afurther embodiment, the bioactive agent is selected from the groupconsisting of proteins, peptides, nucleosides, nucleotides, antiviralagents, antineoplastic agents, antibiotics, and anti-inflammatory drugs.In a further embodiment, the bioactive agent is selected from the groupconsisting of calcitonin, cyclosporin, insulin, oxytocin, tyrosine,enkephalin, tyrotropin releasing hormone (TRH), follicle stimulatinghormone (FSH), luteinizing hormone (LH), vasopressin and vasopressinanalogs, catalase, superoxide dismutase, interleukin-II (IL2),interferon, colony stimulating factor (CSF), tumor necrosis factor (TNF)and melanocyte-stimulating hormone. In a further embodiment, thebioactive agent is an Alzheimer antagonist.

Example No. 16 Nanoparticles with Heparin Core Substrate

Heparin is a negatively charged drug that serves therapeutically asanti-coagulant. Heparin is generally administered by intravenousinjection. Some aspects of the invention relate to heparin nanoparticlesfor oral administration or subcutaneous administration. In a furtherembodiment, heparin serves as at least a portion of the core substratewith chitosan as shell substrate, wherein heparin conjugate at least onebioactive agent as disclosed herein. In preparation, nanoparticles wereobtained upon addition of heparin Leo aqueous solution (2 ml), using apipette (0.5-5 ml, PLASTIBRAND®, BrandTech Scientific Inc., Germany),into a low-MW CS aqueous solution (pH 6.0, 10 ml) with excessconcentrations under magnetic stirring at room temperature.Nanoparticles were collected by ultracentrifugation at 38,000 rpm for 1hour. Table 4 shows the conditions of solution preparation and theaverage nanoparticle size.

TABLE 4 Heparin Chitosan Particle size Conditions conc. @2 ml conc. @10ml (nm) A 200 iu/ml 0.09% 298.2 ± 9.3 B 100 iu/ml 0.09% 229.1 ± 4.5 C 50 iu/ml 0.09% 168.6 ± 1.7 D  25 iu/ml 0.09% 140.1 ± 2.3

To evaluate the pH stability of the heparin-containing nanoparticlesfrom Example no. 16, the nanoparticles from Condition D in Table 4 aresubjected to various pH for 2 hours (sample size=7). Table 5 shows theaverage size, size distribution (polydispersity index: PI) and zetapotential (Zeta) of the nanoparticles at the end of 2 hours undervarious pH environments. The data shows the nanoparticles are relativelystable. In one embodiment, the nanoparticles of the present inventionmay include heparin, heparin sulfate, small molecular weight heparin,and heparin derivatives.

TABLE 5 pH 1.5 2.6 6.6 7.4 Deionized water @5.9 Size (nm) 150 ± 9  160 ±12 153 ± 2  154 ± 4  147 ± 5  PI 0.54 ± 0.03  0.50 ± 0.04 0.08 ± 0.020.32 ± 0.03 0.37 ± 0.02 Zeta (+) 15 ± 2  33 ± 6  15 ± 0.1  11 ± 0.2 18 ±4 

In a further embodiment, a growth factor such as bFGF withpharmaceutically effective amount is added to heparin Leo aqueoussolution before the pipetting step in Example No. 15. In our laboratory,growth factors and proteins with pharmaceutically effective amount havebeen successfully conjugated with heparin to form nanoparticles of thepresent invention with chitosan as the shell substrate, wherein thegrowth factor is selected from the group consisting of VascularEndothelial Growth Factor (VEGF), Vascular Endothelial Growth Factor 2(VEGF2), basic Fibroblast Growth Factor (bFGF), Vascular EndothelialGrowth Factor 121 (VEGF121), Vascular Endothelial Growth Factor 165(VEGF165), Vascular Endothelial Growth Factor 189 (VEGF189), VascularEndothelial Growth Factor 206 (VEGF206), Platelet Derived Growth Factor(PDGF), Platelet Derived Angiogenesis Factor (PDAF), Transforming GrowthFactor-β (TGF-β), Transforming Growth Factor-α (TGF-α), Platelet DerivedEpidermal Growth Factor (PDEGF), Platelet Derived Wound Healing Formula(PDWHF), epidermal growth factor, insulin-like growth factor, acidicFibroblast Growth Factor (aFGF), human growth factor, and combinationsthereof; and the protein is selected from the group consisting ofhaemagglutinin (HBHA), Pleiotrophin, buffalo seminal plasma proteins,and combinations thereof.

In a co-pending application, U.S. patent application Ser. No. 10/916,170filed Aug. 11, 2004, it is disclosed that a biomaterial with free aminogroups of lysine, hydroxylysine, or arginine residues within biologictissues is crosslinkable with genipin, a crosslinker (Biomaterials 1999;20:1759-72). It is also disclosed that the crosslinkable biomaterial maybe crosslinked with a crosslinking agent or with light, such asultraviolet irradiation, wherein the crosslinkable biomaterial may beselected from the group consisting of collagen, gelatin, elastin,chitosan, NOCC (N, O, carboxylmethyl chitosan), fibrin glue, biologicalsealant, and the like. Further, it is disclosed that a crosslinkingagent may be selected from the group consisting of genipin, itsderivatives, analog (for example, aglycon geniposidic acid),stereoisomers and mixtures thereof. In one embodiment, the crosslinkingagent may further be selected from the group consisting of epoxycompounds, dialdehyde starch, glutaraldehyde, formaldehyde, dimethylsuberimidate, carbodiimides, succinimidyls, diisocyanates, acyl azide,reuterin, ultraviolet irradiation, dehydrothermal treatment,tris(hydroxymethyl)phosphine, ascorbate-copper, glucose-lysine andphoto-oxidizers, and the like.

In one embodiment, it is disclosed that loading drug onto achitosan-containing biological material crosslinked with genipin orother crosslinking agent may be used as biocompatible drug carriers fordrug slow-release or sustained release. Several biocompatible plasticpolymers or synthetic polymers have one or more amine group in theirchemical structures, for example poly(amides) or poly(ester amides). Theamine group may become reactive toward a crosslinking agent, such asglutaraldehyde, genipin or epoxy compounds of the present invention. Inone embodiment, the nanoparticles comprised of crosslinkable biomaterialis crosslinked, for example up to about 50% degree or more ofcrosslinking, preferably about 1 to about 20% degree of crosslinking ofthe crosslinkable components of the biomaterial, enabling sustainedbiodegradation of the biomaterial and/or sustained drug release.

By modifying the chitosan structure to alter its charge characteristics,such as grafting the chitosan with methyl, N-trimethyl, alkyl (forexample, ethyl, propyl, butyl, isobutyl, etc.), polyethylene glycol(PEG), or heparin (including low molecular weight heparin, regularmolecular weight heparin, and genetically modified heparin), the surfacecharge density (zeta potential) of the CS-γPGA nanoparticles may becomemore pH resistant or hydrophilic. In one embodiment, the chitosan isgrafted with polyacrylic acid or a polymer with a chemical formula:

By way of illustration, trimethyl chitosan chloride might be used informulating the CS-γPGA nanoparticles for maintaining its sphericalbiostability at a pH lower than pH 2.5, preferably at a pH as low as1.0. Some aspects of the invention provide a drug-loadedchitosan-containing biological material crosslinked with genipin orother crosslinking agent as a biocompatible drug carrier for enhancingbiostability at a pH lower than pH 2.5, preferably within at a pH as lowas 1.0.

Freeze-Dried Nanoparticles

A pharmaceutical composition of nanoparticles of the present inventionmay comprise a first component of at least one bioactive agent, a secondcomponent of chitosan (including regular molecular weight and lowmolecular weight chitosan), and a third component that is negativelycharged. In one embodiment, the second component dominates on a surfaceof the nanoparticle. In another embodiment, the chitosan is N-trimethylchitosan. In still another embodiment, the low molecular weight chitosanhas a molecular weight lower than that of a regular molecular weightchitosan. The nanoparticles may further comprise tripolyphosphate andmagnesium. For example, a first solution of (2 ml 0.1% γ-PGA aqueoussolution @pH 7.4±0.05% Insulin+0.1% Tripolyphosphate (TPP)+0.2% MgSO₄)is added to a base solution (10 ml 0.12% chitosan aqueous solution @pH6.0) as illustrated in Example no. 4 under magnetic stirring at roomtemperature. Nanoparticles were collected by ultracentrifugation at38,000 rpm for 1 hour. Supernatants were discarded and nanoparticleswere resuspended in deionized water for freeze-drying preparation. Otheroperating conditions or other bioactive agent (such as protein, peptide,siRNA, growth factor, the one defined and disclosed herein, and thelike) may also apply.

Several conventional coating compounds that form a protective layer onparticles are used to physically coat or mix with the nanoparticlesbefore a freeze-drying process. The coating compounds may includetrehalose, mannitol, glycerol, and the like. Trehalose, also known asmycose, is an alpha-linked (disaccharide) sugar found extensively butnot abundantly in nature. It can be synthesized by fungi, plants andinvertebrate animals. It is implicated in anhydrobiosis—the ability ofplants and animals to withstand prolonged periods of desiccation. Thesugar is thought to form a gel phase as cells dehydrate, which preventsdisruption of internal cell organelles by effectively splinting them inposition. Rehydration then allows normal cellular activity to resumewithout the major, generally lethal damage, which would normally followa dehydration/rehydration cycle. Trehalose has the added advantage ofbeing an antioxidant.

Trehaloze has a chemical formula as C₁₂H₂₂O₁₁.2H₂O. It is listed as CASno. 99-20-7 and PubChem 7427. The molecular structure for trehalose isshown below.

Trehalose was first isolated from ergot of rye. Trehalose is anon-reducing sugar formed from two glucose units joined by a 1-1 alphabond giving it the name of α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside.The bonding makes trehalose very resistant to acid hydrolysis, andtherefore stable in solution at high temperatures even under acidicconditions. The bonding also keeps non-reducing sugars in closed-ringform, such that the aldehyde or ketone end-groups do not bind to thelysine or arginine residues of proteins (a process called glycation).Trehalose has about 45% the sweetness of sucrose. Trehalose is lesssoluble than sucrose, except at high temperatures (>80° C.). Trehaloseforms a rhomboid crystal as the dihydrate, and has 90% of the calorificcontent of sucrose in that form. Anhydrous forms of trehalose readilyregain moisture to form the dihydrate. Trehalose has also been used inat least one biopharmaceutical formulation, the monoclonal antibodytrastuzumab, marketed as Herceptin. It has a solubility of 68.9 g/100 gH₂O at 20° C.

Mannitol or hexan-1,2,3,4,5,6-hexyl (C₆H₈(OH)₆) is an osmotic diureticagent and a weak renal vasodilator. Chemically, mannitol is a sugaralcohol, or a polyol; it is similar to xylitol or sorbitol. However,mannitol has a tendency to lose a hydrogen ion in aqueous solutions,which causes the solution to become acidic. For this, it is not uncommonto add a substance to adjust its pH, such as sodium bicarbonate.Mannitol has a chemical formula as C₆H₁₄O₆. It is listed as CAS no.69-65-8 and PubChem 453. The molecular structure for mannitol is shownbelow.

Glycerol is a chemical compound with the formula HOCH₂CH(OH)CH₂OH. Thiscolorless, odorless, viscous liquid is widely used in pharmaceuticalformulations. Also commonly called glycerin or glycerine, it is a sugaralcohol and fittingly is sweet-tasting and of low toxicity. Glycerol hasthree hydrophilic alcoholic hydroxyl groups that are responsible for itssolubility in water and its hygroscopic nature. Glycerol has a chemicalformula as C₃H₅(OH)₃. It is listed as CAS no. 56-81-5. The molecularstructure for glycerol is shown below.

Example No. 17 Freeze-Drying Process for Nanoparticles

Nanoparticles (at 2.5% concentration) were mixed with solution from fourtypes of liquid at a 1:1 volume ratio for about 30 minutes until fullydispersed. The mixed particle-liquid was then freeze-dried under alyophilization condition, for example, at −80° C. and <25 mmHg pressurefor about 6 hours. The parameters in a selected lyophilization conditionmay vary slightly from the aforementioned numbers. The four types ofliquid used in the experiment include: (A) DI water; (B) trehalose; (C)mannitol; and (D) glycerol, whereas the concentration of the liquid (A)to liquid (C) in the solution was set at 2.5%, 5% and/or 10%. After afreeze-drying process, the mixed particle-liquid was rehydrated with DIwater at a 1:5 volume ratio to assess the integrity of nanoparticles ineach type of liquid. The results are shown in Table 6. By comparing theparticle size, polydispersity index and zeta-potential data, only thenanoparticles from the freeze-dried particle-trehalose runs (at 2.5%,5%, and 10% concentration level) show comparable properties as comparedto those of the before-lyophilization nanoparticles. Under the same dataanalysis, the nanoparticles from the freeze-dried particle-mannitol runs(at 2.5%, and 5% concentration level) show somewhat comparableproperties as compared to those of the before-lyophilizationnanoparticles.

TABLE 6 Properties of nanoparticles before and after an exemplaryfreeze-drying process. A: DI Water B: Trehalose C: Mannitol D: GlycerolA: DI water + B: Trehalose + C: Mannitol + D: Glycerol + NPs (volume NPs(volume 1:1), NPs (volume 1:1), NPs (volume 1:1), NPs solution 1:1),freeze-dried freeze-dried freeze-dried freeze-dried Conc. 2.50% Conc.Conc. 2.50% 5.00% 10.00% Conc. 2.50% 5.00% Conc. 2.50% 5.00% 10.00% Size266 Size (nm) 9229.1 Size 302.4 316.7 318.9 Size (nm) 420.1 487.5 Size(nm) 6449.1 7790.3 1310.5 (nm) (nm) Kcps 352.2 Kcps 465.3 Kcps 363.7327.7 352.2 Kcps 305.4 303.7 Kcps 796.1 356.1 493.3 PI 0.291 PI 1 PI0.361 0.311 0.266 PI 0.467 0.651 PI 1 1 1 Zeta 25.3 Zeta Zeta 25.6 24.624.7 Zeta 24.4 25.3 Zeta Poten- Potential Poten- Potential Potentialtial tial

FIG. 16 shows an illustrative mechanism of nanoparticles released fromthe enteric-coated capsules. FIG. 16(A) shows the phase of nanoparticlesin the gastric cavity, wherein the freeze-dried nanoparticles 82 areencapsulated within an initial enteric coating or coated capsule 81.FIG. 16(B) shows a schematic of the nanoparticles during the phase ofentering small intestine, wherein the enteric coat and its associatedcapsule starts to dissolve 83 and a portion of nanoparticles 82 isreleased from the capsule and contacts fluid. FIG. 16(C) shows the phaseof nanoparticles in the intestinal tract, wherein the nanoparticlesrevert to a wet state having chitosan at its surface. In an alternateembodiment, nanoparticles may be released from alginate-calcium coating.In preparation, nanoparticles are first suspended in a solution thatcontains calcium chloride, wherein the calcium ions are positivelycharged. With a pipette, alginate with negatively charged carboxylgroups is slowly added to the calcium chloride solution. Under gentlestirring, the alginate-calcium starts to conjugate, gel, and coat on thenanoparticle surface. In simulated oral administration of thealginate-calcium coated nanoparticles, nanoparticles start to separatefrom the coating when they enter the small intestines.

Example No. 18 Freeze-Dried Nanoparticles in Animal Evaluation

In the in vivo study, rats as prepared and conditioned according toExample no. 12 were used in this evaluation. In the animal evaluationstudy, diabetic rats were fasting for 12 hours and subjected to threedifferent conditions: (a) oral deionized water (DI) administration asnegative control; (b) oral insulin-loaded lyophilized nanoparticlesadministration, whereas the nanoparticles have an insulin loadingcontent of 4.4% and an insulin loading efficiency of 48.6% and areloaded in a capsule with surface enteric coating; and (c) subcutaneous(SC) insulin injection at 5 U/kg as positive control. The blood glucoseconcentration from rat's tail was measured over the time in the study.

FIG. 19 shows glucose change (hypoglycemic index) versus time of the invivo animal study (n=5). The glucose change as a percentage of baselines for oral DI administration (control) over a time interval of 10hours appears relatively constant within the experimental measurementerror range. As anticipated, the glucose decrease for the SC insulininjection route appears in rat blood in the very early time interval andstarts to taper off after 2 hours in this exemplary study and ends atabout 6 hours. The most important observation of the study comes fromthe oral administration route with insulin-loaded lyophilized (namely,freeze-dried) nanoparticles. Nanoparticles of this example have insulinLC at 4.4%, whereas nanoparticles from Example no. 12 had insulin LC at14.1% in FIG. 15). With the same amount of nanoparticles in bothexamples, the insulin-feeding ratio of Example no. 18 to Example no. 12is about 1:3. In other words, the insulin fed to a rat in this studyfrom nanoparticles is about ⅓ of the insulin from nanoparticles fed torats in Example no. 12.

The blood glucose begins to decrease from the base line at about 3 hoursafter administration and sustains at a lower glucose level at more than10 hours into study. It implies that the current insulin-loadednanoparticles may modulate the glucose level in animals in a sustainedor prolonged effective mode. Some aspects of the invention provide amethod of treating diabetes of a patient comprising orally administeringinsulin-containing nanoparticles with a dosage effective amount of theinsulin to treat the diabetes, wherein at least a portion of thenanoparticles comprises a positively charged shell substrate and anegatively charged core substrate. In one embodiment, the dosageeffective amount of the insulin to treat the diabetes comprises aninsulin amount of between about 15 units to 45 units per kilogram bodyweight of the patient, preferably 20 to 40 units, and most preferably atabout 25 to 35 units insulin per kilogram body weight. In oneembodiment, the lyophilized nanoparticles may be fed as is to an animalwithout being loaded in an enterically coated capsule.

It is known that Zn (zinc) is usually added in the biosynthesis andstorage of insulin. FIGS. 17 and 18 show a schematic of insulinconjugated with a polyanionic compound (i.e., γ-PGA in this case) via Znand thus increase its loading efficiency and loading content in thenanoparticles of the present invention. It is further demonstrated thatZn may complex with the histidine and glutamic acid residues in insulinto increase the insulin stability and enhance controlled releasecapability or sustained therapy. Some aspects of the invention relate toa nanoparticle characterized by enhancing intestinal or brain bloodparacellular transport, the nanoparticle comprising a first component ofat least one bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein astabilizer is added to complex the at least one bioactive agent to thenegatively charged third component. In one embodiment, the stabilizer iszinc or calcium.

Example No. 19 Nanoparticles with Enhanced Insulin Loading

In a co-pending application, U.S. patent application Ser. No. 11/881,185filed Jul. 26, 2007, entire contents of which are incorporated herein byreference, it is disclosed that a novel nanoparticle may comprise ashell substrate of chitosan and a core substrate consisting of at leastone bioactive agent, MgSO₄, TPP, and a negatively charged substrate thatis neutralized with chitosan in the core. FIG. 20 shows insulin-loadednanoparticles with a core composition comprised of γ-PGA, MgSO₄, sodiumtripolyphosphate (TPP), and insulin. Nanoparticles were obtained uponaddition of core component, using a pipette (0.5-5 ml, PLASTIBRAND®,BrandTech Scientific Inc., Germany), into a CS aqueous solution (pH 6.0,10 ml) at certain concentrations under magnetic stirring at roomtemperature. Nanoparticles were collected by ultracentrifugation at38,000 rpm for 1 hour. Supernatants were discarded and nanoparticleswere resuspended in deionized water for further studies. In oneembodiment, nanoparticles are encapsulated in a gelcap or arelyophilized before being loaded in a gelcap or in a tablet. The sodiumtripolyphosphate has a chemical formula of Na₅P₃O₁₀ as shown below:

In the example, the core composition may be varied and evaluated with apreferred composition of 2 ml γ-PGA aqueous solution at pH 7.4 plusinsulin, MgSO₄ and TPP, resulting in a ratio ofCS:γ-PGA:TPP:MgSO₄:insulin=6.0:1.0:1.0:2.0:0.05. Thus, the nanoparticlesshow characteristics with chitosan shell and a core compositionconsisted of γ-PGA, MgSO₄, TPP, and insulin and have an average loadingefficiency of 72.8% insulin and an average loading content of 21.6%insulin.

In the enhanced drug loading of the present example, there provides twoor more distinct ionic crosslink mechanisms. In one embodiment, thenanoparticles of the present invention may have a structure or matrix ofinterpenetrated ionic-crosslinks (that is, elongate ionic-crosslinkchains) including a first ionic-crosslink chain of NH₃ ⁺ of CS with COO⁻of γ-PGA, a second ionic-crosslink chain of NH₃ ⁺ of CS with SO₄ ²⁻ ofMgSO₄, a third ionic-crosslink chain of Mg²⁺ of MgSO₄ with COO⁻ ofγ-PGA, and/or a fourth ionic-crosslink chain of Na₃P₃O₁₀ ²⁻ of TPP withNH₃ ⁺ of CS or Mg²⁺ of MgSO₄.

Some aspects of the invention relate to a nanoparticle composition fororal administration with the insulin loading efficiency and content athigher than 45% and 14% (preferably up to about 73% and 22%),respectively. The prepared nanoparticles (NPs) are stable in the rangeof pH 2.0 to 7.1. This broad range is to maintain the chitosan-shellednanoparticle transiently stable in most of the intestine region(including duodenum, jejunum, and ileum) for enhanced membraneadsorption and paracellular permeability of active ingredient (forexample, insulin). Some aspects of the invention provide achitosan-shelled nanoparticle with a core composition comprised ofγ-PGA, MgSO₄, TPP, and at least one bioactive agent. In an alternateembodiment, some aspects of the invention provide a chitosan-shellednanoparticle with a core composition consisted of γ-PGA, MgSO₄, TPP, andat least one bioactive agent. In one embodiment, negatively chargedγ-PGA may conveniently be substituted by another negatively chargesubstrate, such as heparin.

Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims. Many modifications and variations are possible inlight of the above disclosure.

1. A method of manufacturing an orally deliverable pharmaceutical composition, the method comprising steps of: (a) providing a drug carrier; (b) providing bioactive nanoparticles, wherein said nanoparticles consist of a positively charged chitosan, a negatively charged substrate, optionally a zero-charge compound, and at least one bioactive agent, wherein a shell portion of said nanoparticles is dominated by said positively charged chitosan; and (c) loading said bioactive nanoparticles in said drug carrier, thereby producing the orally deliverable pharmaceutical composition.
 2. The method of claim 1, wherein said bioactive nanoparticles have a mean particle size between about 50 and 400 nanometers.
 3. The method of claim 1, wherein the drug carrier is a capsule or tablet.
 4. The method of claim 1, wherein the drug carrier is a liquid carrier.
 5. The method of claim 1, further comprising a step of freeze-drying said bioactive nanoparticles prior to the loading step.
 6. The method of claim 5, wherein said bioactive nanoparticles are mixed with a coating compound prior to the freeze-drying step.
 7. The method of claim 6, wherein said coating compound is trehalose or mannitol.
 8. The method of claim 1, wherein said chitosan is N-trimethyl chitosan or a chitosan derivative.
 9. The method of claim 1, wherein said zero-charge compound is a transport enhancer.
 10. The method of claim 9, wherein said at least one transport enhancer is selected from the group consisting of bile salts, anionic surfactants, medium-chain fatty acids, and phosphate esters.
 11. The method of claim 1, wherein said pharmaceutical composition further comprises excipients during the loading step.
 12. The method of claim 1, wherein said nanoparticles are formed via a simple and mild ionic-gelation method.
 13. The method of claim 1, wherein said at least one bioactive agent is a protein or peptide.
 14. The method of claim 1, wherein said at least one bioactive agent is siRNA.
 15. The method of claim 1, wherein said nanoparticles are treated with alginate-calcium coating prior to the loading step.
 16. The method of claim 1, wherein said at least one bioactive agent is insulin or insulin analog.
 17. The method of claim 1, wherein said at least one bioactive agent is a hydrophilic molecule.
 18. The method of claim 1, wherein said at least one bioactive agent is erythropoietin hormone.
 19. The method of claim 1, wherein said at least one bioactive agent is heparin.
 20. The method of claim 1, wherein said at least one bioactive agent is small molecular weight heparin. 